Abstract

Abstract
Background and aims: The aggressiveness of breast cancer is influenced by multiple

cellular and molecular factors, some of which are yet to be elucidated. In normal cells,
vacuolar H (+) -ATPase (V-ATPases), which pump protons across the plasma membrane
function to maintain electrochemical gradient and acidity in intracellular compartments.
Thus, alteration of normal V-ATPase activity could be involved in the modification of
cancerous and tumorigenic processes in breast cancer. The present study investigated the
role of V-ATPase subunit E1 (ATP6V1E1) — the major isoform (77 %) of the five human
ATPases (subunit B, C, D, E, and G) — in the apoptosis, survival, and motility of breast
cancer cells.
Materials and methods: The alteration of the expression of ATP6V1E1 in MCF7 and
MDA-MB-231 cancer cells was achieved using recombinant plasmid DNA carrying full-
length ATP6V1E1 (pcDNA™ 3.1-ATP6V1E1; for upregulation) and RNA interference
(RNAi) technology (siRNA1-4, for downregulation). The cell lines with altered
ATP6V1E1 expression were studied for cellular ATP6V1E1 protein localization using
immunofluorescence confocal microscopy. They were also examined for altered apoptosis
rate using cellular apoptosis assays including Annexin V, Multi-Caspase and Acridine
orange. Cell viability were probed using fluorescence-activated cell sorting (FACS)
analysis. The cells were investigated for intracellular and extracellular acidity (pHi, and
pHe) using pH-sensitive LysoSensor™ Green DND-189 dye (Cat# L7535, Ambion® Life
Techonologies Ltd Paisley, UK) and pH changes of growth media, respectively. Lastly,
the effect of altered ATP6V1E1 expression on actin-tubulin cytoskeleton by
immunofluorescence and electron microscopy and the subsequent influence on cell
motility was investigated. The motility (migration, and invasion) and colony forming
abilities of the cancer cells were investigated using wound scratch closure assay with the
aid of the Image J software. Statistical analysis was performed using GraphPad InStat
software.
Results: Immuno-labelling of the target ATP6V1E1 protein using anti-ATP6V1E1
monoclonal antibody was comparable between two dilutions tested (1:100 and 1:250). A
lower antibody concentration (1:250) was deemed cost-effective. MCF7 and MDA-MB-
231 cells exhibited plasma membrane and cytoplasmic ATP6V1E1 immunolabelling,
respectively. Cytoplasmic microtubules were visualized in both cells, while actin filaments
were clearly visualized in only MCF7 cells. ATP6V1E1 overexpression significantly
decreased cell survival and increased apoptosis rates in both cell lines, compared to
controls (P<0.0001). By contrast, ATP6V1E1 down-expression significantly increased cell
survival, especially cells in S phase and G2/M phase 48 post-plating. ATP6V1E1
overexpression increased actin content in MCF7 cells more than in MDA-MB-231 cells,
which instead revealed higher microtubule labelling than the former. ATP6V1E1
downregulation resulted in faster wound closure rate in both cell lines. The intensity of
LysoSensor™-labelling (pHi) cells was significantly higher in ATP6V1E1-overexpressing
cells compared to controls (p<0.01). Cells grown in the acidic pH (6.5) and alkaline pH
(8.5) exhibited an increased and decreased time-dependent survival from 2 h to 24 h post-
plating compared to controls (p<0.001), respectively. ATP6V1E1 upregulation reduced
actin-tubulin cytoskeleton.
Conclusions: Overexpression of ATP6V1E1 appears to repress breast cancer cell
activities by promoting apoptosis, reducing actin-tubulin cytoskeleton (which reduce
cancer cell motility) and reducing pHi. Recombinant ATP6V1E1 gene products could be
useful for breast cancer therapy warranting further studies.
Keywords: breast cancer, V-ATPases, ATP6V1E1, apoptosis, acidity, motility
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Table of Contents
Abstract………… ............................................................................................................................ i
Acknowledgements ........................................................................... Error! Bookmark not defined.
List of abbreviations .................................................................................................................... 21
CHAPTER 1. Introduction ............................................................. Error! Bookmark not defined.
1.1 Introduction...........................................................................Error! Bookmark not defined.
1.1.1 Pathophysiology and etiology of breast cancer...........Error! Bookmark not defined.
1.1.2 Treatment response of various breast cancer subtypes ............. Error! Bookmark not
defined.
1.1.3 Risk factors for breast cancer ......................................Error! Bookmark not defined.
1.2 The hallmarks of cancer........................................................Error! Bookmark not defined.
1.3 Apoptosis ..............................................................................Error! Bookmark not defined.
1.3.1 Apoptotic pathways and apoptotic mechanisms .........Error! Bookmark not defined.
1.3.2 Genes involved in apoptosis .......................................Error! Bookmark not defined.
1.4 Transmembrane ATPases .....................................................Error! Bookmark not defined.
1.4.1 The structure of vacuolar H (+) -ATPase (V-ATPases) ........... Error! Bookmark not
defined.
1.4.2 V-ATPase function .....................................................Error! Bookmark not defined.
1.4.3 The role of V-ATPase in cancer .................................Error! Bookmark not defined.
1.4.4 Role of V-ATPases in the invasion of MDA-MB-231 breast cancer cells ........Error!
Bookmark not defined.
1.5 Acidic pH levels and cancer .................................................Error! Bookmark not defined.
1.5.1 Cancer cells and the acidic environment.....................Error! Bookmark not defined.
1.5.2 Production and extrusion of metabolic acids ..............Error! Bookmark not defined.
1.5.3 Extracellular pH sensing .............................................Error! Bookmark not defined.
1.5.4 Effect of extracellular acidity......................................Error! Bookmark not defined.
1.5.5 Effect on the cytoskeleton...........................................Error! Bookmark not defined.
1.6 Overarching hypotheses........................................................Error! Bookmark not defined.
1.7 Aims and objectives ..............................................................Error! Bookmark not defined.
1.7.1 Aims ............................................................................Error! Bookmark not defined.
1.7.2 Objectives ...................................................................Error! Bookmark not defined.
CHAPTER 2. Materials and Methods ........................................... Error! Bookmark not defined.
2.1 General cell culture and experiments....................................Error! Bookmark not defined.
2.1.1 Cell culture..................................................................Error! Bookmark not defined.
2.1.2 Freezing and thawing of the breast cancer cell lines ..Error! Bookmark not defined.
2.1.3 Plasmid preparation ....................................................Error! Bookmark not defined.
2.1.4 Plasmid DNA transfection ..........................................Error! Bookmark not defined.
2.1.5 RNA interference by siRNA .......................................Error! Bookmark not defined.
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2.1.6 Optimization of the transfection for the HiPerFect transfection reagent ...........Error!
2.1.7 Cell viability determination and apoptosis detection ..Error! Bookmark not defined.
2.1.7.1 Vital dye exclusion assay ................................... Error! Bookmark not defined.
2.1.7.2 Cell viability using flow cytometry ..................... Error! Bookmark not defined.
2.1.7.3 Determination of apoptosis using flow cytometry ............ Error! Bookmark not
defined.
2.1.8 Cell cycle analysis ......................................................Error! Bookmark not defined.
2.1.9 Anchorage-dependent clonogenic assay .....................Error! Bookmark not defined.
2.1.10 Cell migration .............................................................Error! Bookmark not defined.
2.2.11 Fluorescent staining of cultures ..................................Error! Bookmark not defined.
Labelling (Antibodies)........................................................ Error! Bookmark not defined.
Antibody preparation ......................................................... Error! Bookmark not defined.
Mounting ............................................................................ Error! Bookmark not defined.
2.2.12 Ultrastructural analysis of breast cancer cells .............Error! Bookmark not defined.
2.2.12.1 Sample preparation for Transmission electron microscopy (TEM) ....... Error!
2.2.12.2 Staining ultrathin sections.............................. Error! Bookmark not defined.
2.2.12.3 Microscopy ..................................................... Error! Bookmark not defined.
2.2.13 Effects of ATP6V1E1 over-expression and down regulation on the intracellular and
extracellular pH in MCF7 and MDA-MB-231 cells ........Error! Bookmark not defined.
2.2.14 Effects of altering extracellular pH on the survival of breast cancer cells .........Error!
2.2.15 Statistical analysis .......................................................Error! Bookmark not defined.
CHAPTER 3. Characterization of breast cancer cell lines by immunocytochemistry and
electron microscopy................................................. Error! Bookmark not defined.
3.3 Materials and Methods .........................................................Error! Bookmark not defined.
3.4 Results ..................................................................................Error! Bookmark not defined.
3.4.1 Optimization of Anti-ATP6V1E1 monoclonal antibody labelling .. Error! Bookmark
not defined.
3.4.2 ATP6V1E1 protein localization in MCF7 and MDA-MB-231 cancer cells......Error!
3.4.3 Quantitative analysis of the immunofluorescence staining intensity .................Error!
3.4.4 Expression of the ATP6V1E1 protein by fluorescence intensity histogram......Error!
3.4.5 Ultrastructural analysis of MDA-MB-231 and MCF7 cells ..... Error! Bookmark not
defined.
3.5 Discussion...........................................................................Error! Bookmark not defined.
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3.6 Conclusion ..........................................................................Error! Bookmark not defined.
CHAPTER 4. Effect of ATP6V1E1 expression levels on basal apoptosis of MCF7 and
MDA-MB-231 breast cancer cells .......................... Error! Bookmark not defined.
4.3 Materials and methods ..........................................................Error! Bookmark not defined.
4.3.1 Methods ......................................................................Error! Bookmark not defined.
4.4.1 ATP6V1E1-labelling intensity in over/down-expressing MCF7 and MDA-MB-231
cells ..................................................................................Error! Bookmark not defined.
4.4.2 Effect of ATP6V1E1 over-expression on ATP6V1E1 intensity in MCF7 and
MDA-MB-231 cells .........................................................Error! Bookmark not defined.
4.4.3 Effect of ATP6V1E1 depletion on its intensity and expression in MCF7 and MDA-
MB-231 cells....................................................................Error! Bookmark not defined.
4.4.4 The effects of ATP6V1E1 over-expression on MCF7 breast cancer cells ........Error!
4.4.5 Effect of ATP6V1E1 over-expression on spontaneous apoptosis and long-term
survival of MCF7 breast cancer cells. ..............................Error! Bookmark not defined.
4.4.6 Effects of ATP6V1E1 over-expression on the survival of metastatic breast cancer
cells MDA-MB231 ..........................................................Error! Bookmark not defined.
4.4.7 The effects of ATP6V1E1 down-regulation on breast cancer cell lines ............Error!
4.4.8 The effects of ATP6V1E1 downregulation in MDA-MB231 breast cancer
cells ..................................................................................Error! Bookmark not defined.
4.4.9 The effect of pcDNA3.1 and ATP6V1E1 on the ultrastructure of MCF7 cells .Error!
4.4.10 The effect of ATP6V1E1 on the ultra-structure of MDA-MB-231 cells ...........Error!
4.5 Discussion .............................................................................Error! Bookmark not defined.
4.6 Conclusion ............................................................................Error! Bookmark not defined.
CHAPTER 5. The role of the ATP6V1E1 in motility of breast cancer cells .. Error! Bookmark
not defined.
5.2 Materials and Methods .........................................................Error! Bookmark not defined.
5.3.1 Effect of upregulation of ATP6V1E1 protein on actin protein in MCF7 and MDA-
MB-231 cancer cells ........................................................Error! Bookmark not defined.
5.3.2 Effect of upregulation of ATP6V1E1 protein on tubulin in MCF7 and MDA-MB-
231 cancer cells ................................................................Error! Bookmark not defined.
5.3.3 Effect of down-regulation of ATP6V1E1 protein on motility (migration) of MCF7
and MDA-MB-231 cancer cells. ......................................Error! Bookmark not defined.
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CHAPTER 6. The effect of ATP6V1E1-mediated alteration of pHi and pHe on the survival
of MCF7 and MDA-MB-231 cells .......................... Error! Bookmark not defined.
6.2.1 Aims ............................................................................Error! Bookmark not defined.
6.2.2 Objectives ...................................................................Error! Bookmark not defined.
6.3 Materials and methods ..........................................................Error! Bookmark not defined.
6.4.1 Effect of ATP6V1E1-overexpression on intracellular acidity .. Error! Bookmark not
defined.
6.4.2 Effect of ATP6V1E1-down regulation on intracellular acidity Error! Bookmark not
defined.
6.4.3 Effect of extracellular pH on the survival breast cancer cell lines ... Error! Bookmark
not defined.
6.4.4 Monitoring of acidity in growth medium....................Error! Bookmark not defined.
6.4.5 Effect of RPMI medium acidification on the intensity of ATP6V1E1 and tubulin in
cancer cells .......................................................................Error! Bookmark not defined.
CHAPTER 7. General Discussion and Conclusion....................... Error! Bookmark not defined.
7.1 V-ATPase localization ..........................................................Error! Bookmark not defined.
7.2 V-ATPase, cytoskeletal reorganization, and metastatic potential ...... Error! Bookmark not
defined.
7.3 V-ATPase, cellular acidity and metastaticity potential.........Error! Bookmark not defined.
7.4 V-ATPase and sensitivity of cancer cells to chemotherapeutic drugs Error! Bookmark not
defined.
References……… .............................................................................. Error! Bookmark not defined.
vi
List of figures
Figure 1.1: The risk for breast cancer are multifactorial with increasing age and family history
being the primary risk factors (Winchester, 2006). Error! Bookmark not defined.
Figure 1.2: The hallmarks of oncogenesis include cell death resistance, sustained proliferation
signaling, evasion of growth suppressors, activation of invasiveness and metastasis,
sustaining replicative immortality and induction of angiogenesis (Hanahan &
Weinberg, 2011). .................................................... Error! Bookmark not defined.
Figure 1.3: Cell apoptosis versus necrosis; unlike cellular necrosis in which cellular contents
including organelles are nonfunctional after cell rapture, cellular apoptosis often
produces apoptotic bodies containing functional organelles as well as DNA
materials. Thus, unlike cellular necrosis, apoptosis does not induce inflammatory
responses; though aberrant apoptosis can produce aberrant DNA materials, which
are oncogenic in nature (Bhat et al., 2015). ............ Error! Bookmark not defined.
Figure 1.4: The intrinsic and extrinsic apoptotic pathways. Apoptosis induction via the death
receptor can result in activation of the extrinsic pathway. Intrinsic pathway can be
initiated as a result of DNA damage or exposure to chemotherapy or radiotherapy
(De Vries et al., 2006). Both involve a cascade of molecular signaling events that
result in controlled destruction of the cell............... Error! Bookmark not defined.
Figure 1.5: The intrinsic and extrinsic pathways of apoptosis. The intrinsic pathway is driven by
proapoptotic factors, which are triggered by irreparable DNA damage leading to the
Bax-mediated perforation of the mitochondrial membrane to aid in the release of
cytochrome C. Cytochrome C together with procaspase-9 and Apaf-1 form the
apoptosome, which activates caspase-9, which in turn activates caspase-3 which
initiates apoptotic degradation of the abnormal cell. By contrast, in the extrinsic
pathway, caspase-8 is activated upon lethal ligands binding to specific receptors and
subsequent formation of the Disc complex (Favaloro et al., 2012). ............... Error!
Figure 1.6: p53 activity in apoptosis. p53 is a tumor suppressor protein, which induces the intrinsic
apoptosis pathway by activating Bax and PUMA. . Error! Bookmark not defined.
Figure 1.7: A diagram showing the structure of V-ATPase. V-ATPase is composed of V1 and V0
domains. The V1 domain is composed of A and B subunits, three copies of E and G
subunits, one copy of C and H subunits and one copy of subunits D and F. The V0
section includes a ring of subunits (c, c' and c") and a, e subunits (Sun-Wada et al.,
2015). ...................................................................... Error! Bookmark not defined.
Figure 1.8: The different subunits of human V-ATPase domains, the cytosolic catalytic domain V1
responsible for ATP hydrolysis and the integral domain V0 responsible for proton
translocation across the membrane. The genes and functions of the subunits are also
shown. ..................................................................... Error! Bookmark not defined.
Figure 1.9: A diagram indicating the role of V-ATPase in cancer. (1) During glycolysis (green
circles represent V0 and purple circles represent V1), the protons generated are
pumped out of the cells by the action of membrane V-ATPase. This will result in the
slight alkalinization of cytoplasm which leads to increases in cell proliferation and
protection from apoptosis; (2) Intracellular V-ATPase functions to acidify secretory
vesicles which is essential for protease secretion and activation. The interaction
between V-ATPase and actin (pink wave) leads to the translocation of V-ATPase to
the plasma membrane. The accumulation of V-ATPase on the plasma membrane
together with the extracellular acidic environment and active proteases will facilitate
metastasis and cell migration; (3) V-ATPase affects signaling pathways by
vii
facilitating the release of ligands from receptors promoting receptor recycling; (4)
V-ATPases promote anticancer drug resistance. Inhibition of V-ATPase has been
reported to increase chemotherapy-induced apoptosis that could be the result of an
increase in influx of the drugs because of the alkaline extracellular conditions as a
result of V-ATPase inhibition (Lu & Qin, 2012). ... Error! Bookmark not defined.
Figure 1.10: Role of V-ATPases in the invasion of breast cancer cells (Cotter et al., 2015). Error!
Figure 1.11: Acidity (H+) regulation in cancer cells. A number of proteins and chemical reactions
detect and regulate pH in cells. NHE1 and V-ATPase. 1: CAs, 2: ATP-Synthase, 3:
NHE1, 4: MCTs, 5: V-H + -ATPase, 6: Cl − /HCO 3 − . pHi = Intracellular pH.
pHe = Extracellular pH (Walsh et al., 2015)........... Error! Bookmark not defined.
Figure 1.12: Construction of a pH-sensor device. G-protein-coupled with receptor (TDAG8)

subsequently senses extracellular proton levels and initiates the balancing process
(Ausländer et al., 2014). ......................................... Error! Bookmark not defined.
Figure 2.1: Nucleofector 2B Device is a single cuvette-based system, which allows efficient
transfection of hard-to-transfect cell lines and primary cells with different substrates
(e.g., Plasmid DNA vectors or siRNA oligonucleotides) in low-throughput format
(Lonza Biosciences). ............................................... Error! Bookmark not defined.
Figure 2.2: The Transfection efficiency of the MCF7 using Nucleofection Transfection Method.
Representative images showing transfected MCF7 cells expressing GFP using
Nucleofection Transfection Method. Transfection efficiency 48h post transfection
was 77±10% ............................................................ Error! Bookmark not defined.
Figure 2.3: Efficient MDA-MB-231 transfection using RNAifect. Representative images showing
the high transfection efficiency obtained when using RNAifect to transfect MDA-
MB-231. Transfection efficiency at 72 h post transfection was 75%. ............ Error!
Figure 2.4: Neubauer hemocytometer. Average density of cells in four large squares X dilution
factor X 104 cells/ mL.. Neubauer hemocytometer above is an enclosed chamber that
has two ports for sample introduction. In the chamber, there are precisely spaced
lines arranged in a grid pattern. In the chamber, there are two counting areas for
replicates (A). The diagram also shows the count chamber of the hemocytometer
indicating the set of 16 squares which should be utilized for counting (B) (Avelar-
Freitas et al., 2014). ................................................ Error! Bookmark not defined.
Figure 2.5: Muse output showing results of the number of viable cells and viability. These results
were arrived at after completion of acquisition utilizing the Muse™ Cell Count and
Viability Software that performs calculations and displays data in two dot plots
automatically. .......................................................... Error! Bookmark not defined.
Figure 2.6: An exhibition of the ratio of dead and apoptotic cells after completion of data attainment
by Muse. The Muse™ Annexin V & dead cell Software instantaneously performs
calculations and displays data in two dot plots. ...... Error! Bookmark not defined.
Figure 2.7: The percentage of cells in different cell cycle phase display using the Muse. After data
acquisition is complete, the Muse™ cell cycle software instantaneously calculates
and displays data in histogram and dot plots. ......... Error! Bookmark not defined.
Figure 3.1: Immunocytochemistry of the ATP6V1E1 protein in PFA-fixed MCF7 breast cancer
cell line stained with anti-ATP6V1E1 Monoclonal antibody at dilution 1:100 (A, B)
and dilution 1:250 (C, D). Panels (B and D), show differential interference contrast
viii
(DIC) counterparts of panels (A) and (C), respectively. Immunolabelling was
principally concentrated in the periphery (probably plasma membrane) of the cells
as opposed to cytoplasm. A 1:100 antibody dilution resulted comparable degree of
immuno-labelling (A), to that of 1:250 (B) but the latter was fainter. Scale bar = 150
μm. .......................................................................... Error! Bookmark not defined.
Figure 3.2: Immunocytochemistry analysis of ATP6V1E1 protein in the PFA-fixed MDA-MB-231

breast cancer cell line stained with anti-ATP6V1E1 Monoclonal antibody at
dilutions 1:100 (A, B) and 1:250 (C, D). In general, immunolabelling was distributed
in cytoplasm, rather than plasma membrane, indicating evidence of vacuolar
localization of ATP6V1E1 protein. Panels (B) and (D), show the DIC counterpart
of panels (A) and (C), respectively. These cells exhibited a predominant cytoplasmic
labelling with little plasma membrane labelling. Scale bar = 150 μm. ........... Error!
Figure 3.3: Immunocytochemical analysis of the ATP6V1E1 protein in PTA-PFA-fixed MCF7

cells (A, B) (Scale bar, 150 μm) and MDA-MB-231 cells (C, D) (Scale bar, 100
μm). stained with anti-ATP6V1E1 monoclonal antibody at dilution 1:250. Panels
(B) and (D), show the phase contrast counterparts of panels (A) and (C),
respectively. Vacuoles can be seen in MDA-MB-231 cells, but not MCF7 cells.
Vacuoles are clearly seen in C, while D requires some contrast enhancement to
reveal the vacuoles. ................................................. Error! Bookmark not defined.
Figure 3.4: Immunocytochemical comparison of the ATP6V1E1 protein at antibody dilution 1:250
in PFA-fixed MCF7 breast cancer cell line (A, B) and PFA-fixed MDA-MB-231
cells (C, D). Labelling and imaging conditions for both cell lines were identical.
Panels (B) and (D), show the DIC counterparts of panels (A) and (C), respectively.
By visual inspection, MCF7 cells (A, B) exhibited significantly greater
immunolabelling intensity compared to that of the MDA-MB-231 cells (C, D). This
is an indication of higher levels of ATP6V1E1 protein and higher number of
vacuoles in MCF7 cells than MDA-MB-231 cells. Scale bar = 150 μm. ....... Error!
Figure 3.5: Immunofluorescence staining intensity of ATP6V1E1 protein marker on MCF7 and
MDA-MB-231 cancer cell lines immuno-labelled with anti-ATP6V1E1 Monoclonal
antibody (1:250). MCF7 cells exhibited significantly higher mean intensity (>60)
compared to a mean intensity of ~20 for MDA-MB-231 cells indicating high levels
of ATP6V1E1 protein, higher number of vacuoles (confirmed by visual inspection)
in MCF7 cells than MDA-MB-231 cells (n=20) (p<0.001).Error! Bookmark not
defined.
Figure 3.6: The two cancer cell lines are shown, with the peripheral labelling in MCF7 cell lines
(A) and cytoplasmic labelling in the MDA-MB-231 cells (C). The profile graphs
represent the fluorescence intensity across the lines drawn on each image. The
expression level of the ATP6V1E1 protein was higher in the MCF7 cells (B) than in
MDA-MB-231 cells (D), evidenced by 4 peaks of labeling (blue arrows) in MCF7
cells (B) compared to MDA-MB-231 cells, which shows only 2 peaks of labeling
(D), which indicates vascular labelling). Red arrows indicate cell profile Scale bar
= 150 μm. ................................................................ Error! Bookmark not defined.
Figure 3.7: Selected area of Figure 3.3 showing contrast enhancement of the vacuole. Labelling is
more intense around the periphery of the vacuole than in the adjacent cytoplasm.
Scale bar = 100 μm. ................................................ Error! Bookmark not defined.
Figure 3.8: The Aclar stripping technique (A) provides a high success rate in allowing numerous
cells to be evaluated in one section. Scale bars = 10 μm. Ultrastructure of MCF7 and
MDA-MB-231 cells (C and D, respectively). ......... Error! Bookmark not defined.
ix
Figure 3.9: Ultrastructure of MCF7 and MDA-MB-231 cells (A and B, respectively). The images
show organelles such as vacuoles (black arrow) and nucleus (blue arrows). Actin
cables can be seen in MCF7 cells running through the cytoplasm (green arrows).
Vacuoles can be seen in both MCF7 cells and to MDA-MB-231 cells, but they
appear to be larger in the MCF7 cells. Scale bars = 5 μm.Error! Bookmark not
defined.
Figure 3.10: TEM of cytoplasm of several MCF7 cells showing numerous cytoplasmic vacuoles
(black arrow) along with actin cables (red arrows). Scale bars = 5 μm. ......... Error!
Figure 3.11: TEM of cytoplasm of two MDA-MB-231 cells showing irregular nuclei (blue arrows)
and nucleolus (red arrow). Scale bars = 2 (A) and 5 (B) µm.Error! Bookmark not
defined.
Figure 3.12: TEM of MCF7 cells showing that: A) a moderate population of microtubules (red
arrows) and (B) abundant actin fibers can be seen (blue arrows); The actin
fibers/cables (Green arrow) frequently form circular structures and these can
surround and encircle vacuolar structures (black arrow). Scale bars = 0.5 µm.
................................................................................ Error! Bookmark not defined.
Figure 3.13: TEM of MCF7 cells showing: A) close up of the vacuole shown in Figure 3.11B
(black arrow) surrounded by bundles of actin fibers (Green arrows). The fibers
appear extremely close to the vacuolar membrane; B) mitochondria (yellow arrows)
surrounded by actin fibers/cables in close proximity (Green arrow). Scale bars = 0.5
µm. .......................................................................... Error! Bookmark not defined.
Figure 3.14: TEM of MCF7 showing: A) desmosome (red arrow) and bundles of actin fibers (Green
arrow); B) junctional complex including desmosomes (red arrow), adherens (blue
arrow) and potentially tight junctions (red arrow), actin fibers in bundles (green
arrows). Scale bars = 0.5 µm. ................................. Error! Bookmark not defined.
Figure 3.15: TEM of MCF7 showing: A) fatty bodies potentially lipid droplets (black arrows), with
a peripheral crescent of material and denser core, microtubules (red arrows) and
bundles of actin fibers (green arrows); B shows bundles of actin fibers (green
arrows) running through the cytoplasm, concentrated towards the cell boundary
(blue arrows). Scale bars = 0.5 µm. ........................ Error! Bookmark not defined.
Figure 3.16: TEM of the cytoplasm of MDA-MB-231 cells showing mitochondria (yellow arrows),
microtubules (red arrows) and small, irregular vacuoles (black arrows). Grey/black
inclusion bodies can also be observed (green arrows). Scale bars = 0.5 µm. . Error!
Figure 3.17: Figure 3.18: Figure 3.18: TEM of the cytoplasm of MDA-MB-231 cells showing
mitochondria (yellow arrows), microtubules (red arrows) and small, irregular
vacuoles (black arrows). Black inclusion bodies can also be observed (green
arrows). Scale bars = 0.5 µm. ................................. Error! Bookmark not defined.
Figure 4.1: ATP6V1E1 over-expression in MCF7 was achieved using pcDNA3.1-V-ATP6V1E1

plasmid. The overexpression was marked by an increased intensity (brighter) of the
ATP6V1E1 labelling. The labelling was predominantly localized to the plasma
membrane. The right panels represent differential interference contrast (DIC)
microscopy images requires contrast enhancement for better viewing. Scale bars =
200 μm. ................................................................... Error! Bookmark not defined.
Figure 4.2: ATP6V1E1 over-expression in MDA-MB-231 cells was achieved using pcDNA3.1-v-
ATP6V1E1 plasmid. The overexpression was marked by an increased intensity
x
(brighter) of the ATP6V1E1 labelling. The labelling was predominantly
cytoplasmic. The lower panels are DIC images of the same cells. requiring contrast
enhancement for better viewing. Scale bars = 200 μm.Error! Bookmark not
defined.
Figure 4.3: ATP6V1E1 downregulation in the MCF7 cells was achieved using ATP6V1E1 specific
siRNAs (1-4). Deregulation was marked by a diminished intensity (reduced
brightness) of the ATP6V1E1 labelling. The labelling was, however, predominantly
localized to the plasma membrane. The lower panels are DIC images of the same
cells. Scale bars = 200 μm. .................................... Error! Bookmark not defined.
Figure 4.4: ATP6V1E1 downregulation in the MDA-MB-231 cells was achieved using ATP6V1E1
specific siRNAs (1-4). Downregulation was marked by a diminished intensity
(reduced brightness) of the ATP6V1E1 labelling. The labelling was, however,
predominantly localized to the plasma membrane. The lower panels are DIC images
of the same cells. Scale bars = 200 μm. ................. Error! Bookmark not defined.
Figure 4.5: The ATP6V1E1 labeling intensities in MCF7 and MDA-MB-231 cells upregulated
with PcDNA3.1- ATP6V1E1; A) In MCF7 cells, the intensities were ~40, 13 and
12, for ATP6V1E1-treated, PcDNA3.1-treated and control cells, respectively. The
difference was statistically significant (n=10) (p<0.001); B) In MDA-MB-231 cells,
the intensities were ~17, 7 and 5, for ATP6V1E1-treated, PcDNA3.1-treated and
control cells, respectively. The difference was statistically significant (n=10)
(p<0.0001). The intensities in MCF7 cells were generally higher compared to MDA-
MB-231 cells........................................................... Error! Bookmark not defined.
Figure 4.6: ATP6V1E1 expression profiles of MCF7 and MDA-MB-231 cells down-regulated with
ATP6V1E1-siRNA1, 2 & 4; A) In MCF7 cells, the degrees of expression were ~130,
~70, ~250, and 260 for ATP6V1E1-siRNA4, ATP6V1E1-siRNA2, negative siRNA,
and control, respectively. The expression difference between ATP6V1E1-siRNA2,
4 and controls was statistically significant (p<0.0001). The downregulation effect
was higher in siRNA2 than siRNA4; B) In MDA-MB-231 cells, the degrees of
expression were ~130, ~140, ~240, and ~225, for siRNA4, siRNA1, negative
siRNA, and control, respectively (p<0.0001). ........ Error! Bookmark not defined.
Figure 4.7: The intensities of MCF7 and MDA-MB-231 cells down-regulated with ATP6V1E1-
siRNA1, 2 & 4; A) In MCF7 cells, the intensities were ~42, ~13 and 17, for
ATP6V1E1-siRNA2, negative siRNA, and control, respectively. The intensity
differences were statistically significant (p<0.001). In MDA-MB-231 cells, the
intensities were ~32, ~30, ~21, and ~17, respectively. ATP6V1E1-siRNA4,
ATP6V1E1-siRNA1, negative siRNA, and control, respectively. The intensity
differences were also significant (p<0.05). ............. Error! Bookmark not defined.
Figure 4.8: Effects of ATP6V1E1 transfection on the expression of endogenous ATP6V1E1 in

MCF7 cells. Western blotting technique shows a highly significant elevation in the
expression of ATP6V1E1 gene in transfected cells as compared to control
(P<0.0001). A) Immunoblot of ATP6V1E1 protein expression in MCF7-pcDNA3.1-
ATP6V1E1-transfected cells, MCF7-pcDNA3.1-transfected cells and MCF7
parental cells. Each lane contains 40 μL of whole-cell lysate subjected to SDS-
polyacrylamide gel electrophoresis (PAGE), followed by Western blot analysis with
anti- ATP6V1E1 antibody. β-actin was used as a control. A Scale bar graph
represents mean ± SEM from 6 independent experiments. Relative expression is the
ratio of ATP6V1E1 level versus βactin. The figure shows about a four-fold increase
in the gene expression in the cells transfected with pcDNA3.1- ATP6V1E1 as
compared to the control cells and pcDNA3.1 transfected cells (n=3). ........... Error!
xi
Figure 4.9: Effects ATP6V1E1 over-expression on total cell number. Total cell number was
determined by fluorescence-activated cell sorting (FACS) analysis. The results show
a significant reduction in total cell count in MCF7 cells transfected with pcDNA3.1-
ATP6V1E1 after 48 hours of the transfection in comparison to the control and
pcDNA3.1 (n=3) (P<0.05). ..................................... Error! Bookmark not defined.
Figure 4.10: ATP6V1E1 over-expression in MCF7 breast cancer cells decreases the viable cell
number at 72 h post-transfection. A) Viable cell count determined by FACS analysis
show a significant reduction in viable cell count in MCF7 cells transfected with
pcDNA3.1- ATP6V1E1 after 72 h of the transfection in comparison to the control
and pcDNA3.1(n=3) (P<0.05). B) Similarly, viable cell count by vital dye stain
method shows a reduction in viable cell count of MCF7 cells with ATP6V1E1
overexpression 72 h after transfection as compared to the control and
pcDNA3.1(n=3) (*P<0.05). .................................... Error! Bookmark not defined.
Figure 4.11: The effect of ATP6V1E1 over-expression on apoptosis in MCF7 cells. MCF7 cells
were transfected with empty vector pcDNA3.1 and pcDNA3.1-ATP6V1E1; where
the apoptosis levels were measured after 24 h. ATP6V1E1 up-regulation in MCF7
cells resulted in an increase in the percentage of total apoptosis. The percentage of
total apoptosis was higher in cells transfected with pcDNA3.1-ATP6V1E1 than
controls (P<0.0001). Apoptosis was measured using Muse annexin V & dead cell
assay (n=3). ............................................................. Error! Bookmark not defined.
Figure 4.12: The effect of ATP6V1E1 over-expression on apoptosis. MCF7 cells were transfected
with empty vector pcDNA3.1 and pcDNA3.1- ATP6V1E1. and apoptosis levels
measured at 24 and 48 h post-plating. A) At 24 h, apoptosis percentage using
acridine orange stain was significantly higher in MCF7 cells up-regulated with
ATP6V1E1 as compared to the controls (*P<0.05). B) At 48 h post-plating, the
percentage of total apoptotic cells was also significantly higher in ATP6V1E1
transfected cells as compared to control (*P<0.05). A reduction in the level of
apoptosis was noticed at 48 h post-plating (n=3).... Error! Bookmark not defined.
Figure 4.13: The effect of ATP6V1E1 over-expression on apoptosis rates: MCF7 cells were
transfected with empty vector pcDNA3.1 and pcDNA3.1-ATP6V1E1 or none
(control) and apoptosis rates measured after 24 h and 48 h post-plating using Muse®
MultiCaspase Assay Kit. A) At 24 h ATP6V1E1-tranfected cells exhibited
significantly higher (>2-fold increase) apoptosis rates as compared to the controls
(p<0.001). B) Although apoptosis rates reduced by two-fold in all cells at 48 h,
apoptosis rate in ATP6V1E1-tranfected cells remained 2-fold higher than controls
(n=3). ...................................................................... Error! Bookmark not defined.
Figure 4.14: A) ATP6V1E1 over-expression decreased the ability of MCF7 breast cancer cells to
survive and form colonies. Clonogenic assay demonstrates that long-term survival
of the cells is compromised after transfection with ATP6V1E1 constructs (n=3). B)
An example image of clonogenic assay plate after crystal violet staining (n=3) versus
cells transfected with pcDNA3.1 alone (*P<0.05). Error! Bookmark not defined.
Figure 4.15: ATP6V1E1 overexpression altered cell-cycle profile of MCF7 cells analyzed 48 h
post-plating. Cell-cycle analysis revealed a significant elevation in the number of
the pcDNA3.1-ATP6V1E1 transfected cells in sub G0 and G0/G1 phase compared
to cells transfected with pcDNA3.1 alone (P<0.0001), This associated with a
significant reduction in the number of the transfected cells in S and G2/M phases
(P<0.0001) as compared to pcDNA3.1 (n=3). ........ Error! Bookmark not defined.
Figure 4.16: Effects of ATP6V1E1 transfection in MDA-MB231 cells. A) Immunoblot of

ATP6V1E1 protein expression in MDA-MB231transfected with pcDNA3.1, MDA-
MB231 transfected ATP6V1E1 and MDAMB231 parental cells was determined at
xii
24 h post-plating. Each lane contains 40 μL of whole-cell lysate subjected to SDS
polyacrylamide gel electrophoresis (PAGE), followed by Western blot analysis with
anti-ATP6V1E1 antibody. β-actin was used as a control. B) Western blotting
technique shows a highly significant elevation in the expression of ATP6V1E1 gene
in transfected cells as compared to control (*P<0.05). A Scale bar graph represents
mean ± SEM from one independent experiment. The figure shows about five-fold
increase in the gene expression in the cells transfected with pcDNA3.1- ATP6V1E1
as compared to the controls and pcDNA3.1 transfected cells (n=3). (Relative
expression is the ratio of ATP6V1E1 level versus β-actin).Error! Bookmark not
defined.
Figure 4.17: Total number of cells and cell viability in ATP6V1E1 over-expressing MDA-MB231
cells compared to controls. Total cell count was determined at 24 h while viability
was determined 48 h and 72 h post-plating using FACS analysis (n=3). A) A
significantly lower total cell count was observed in MDA-MB231 cells transfected
with ATP6V1E1 compared to controls (*P<0.01). B) Viable cell count was
significantly reduced in ATP6V1E1 over-expressing MDA-MB231 cells after 48 h
of transfection compared to the controls (n=3) (*P<0.001). C) After 72 h, there was
a further reduction in % viability in comparison to the control (*P<0.001). .. Error!
Figure 4.18: ATP6V1E1 over-expression in MDA-MB231 cell line decreased viable cell count.
MDA-MB231cells were transfected with empty vector pcDNA3.1 and pcDNA3.1-
ATP6V1E1. Viable cell counts were determined 48 h and 72 h post transfection
(n=3). A) Viable cell count was determined by FACS analysis. The results show a
significant reduction in viable cell count in MDA-MB231 cells transfected with
pcDNA3.1-ATP6V1E1 after 48 h of the transaction in comparison to the control
(*P<0.0001) and pcDNA3.1 (P<0.05). B) Viable cell count by vital dye stain shows
a reduction in viable cell count of MDA-MB231 cells with ATP6V1E1
overexpression 72 h after transfection as compared to the control and pcDNA3.1
(n=3) (*P<0.0001). ................................................. Error! Bookmark not defined.
Figure 4.19: Effects of ATP6V1E1 over-expression on MDA-MB231 breast cancer cells. Cells
were nucleofected with ATP6V1E1 or empty vector as a control (n=3) and viable
cell count determined by FACS analysis. A) The graph shows further reduction in
viability after 72 h of the transfection in comparison to the control (*P<0.001). B)
Results show a further reduction in viable cell count in the MDA-MB231 cells
transfected with ATP6V1E1 after 72 h of the transfection in comparison to the
control (*P<0.01 ..................................................... Error! Bookmark not defined.
Figure 4.20: The effect of ATP6V1E1 over-expression on apoptosis. MDA-MB231 cells were
transfected with empty vector pcDNA3.1 and pcDNA3.1- ATP6V1E1 and apoptosis
levels were measured at different time points. A) ATP6V1E1 up-regulation in
MDA-MB231 cells increased the percentage of total apoptosis. The figure shows
that the percentage of total apoptosis is higher in the cells transfected with
pcDNA3.1-ATP6V1E1 than that in the control and pcDNA3.1 transfected cells after
24 h of the plating (*P<0.05). Apoptosis was measured using FACS analysis. B)
The percentage of total apoptotic cells were also significantly higher in ATP6V1E1
transfected cell as compared to control (*P<0.05) a reduction in the level of
apoptosis were noticed at 48 h post-plating (n=3). . Error! Bookmark not defined.
Figure 4.21: Representative images of cells from three independent experiments were shown.
Acridine orange staining to distinguish between normal and apoptotic cells.
Condensed or fragmented nucleus with indicates apoptosis.Error! Bookmark not
defined.
xiii
Figure 4.22: The effect of ATP6V1E1 over-expression on apoptosis. MDA-MB231 cells were
transfected with empty vector pcDNA3.1 and pcDNA3.1-ATP6V1E1 24 h post-
plating, apoptosis percentage using acridine orange stain was significantly higher in
MDA-MB231 cells up-regulated with ATP6V1E1 as compared to the control and
pcDNA3.1 (P<0.0001). ........................................... Error! Bookmark not defined.
Figure 4.23: The effect of ATP6V1E1 over-expression on apoptosis rates: MDA-MB231 cells
were transfected with empty vector pcDNA3.1 and pcDNA3.1-ATP6V1E1 or none
(control) and apoptosis rates measured after 24 h and 48 h post-plating using Muse®
MultiCaspase Assay Kit. A) ATP6V1E1-tranfected cells exhibited significantly
higher (>2-fold increase) apoptosis rates as compared to the controls (p<0.001). B)
Although, in general, the apoptosis rates reduced slightly in all cells at 48 h post-
plating, apoptosis rate in ATP6V1E1-tranfected cells remained 2-fold higher than
controls (n=3).......................................................... Error! Bookmark not defined.
Figure 4.24: Survival of MDA-MB231 breast cancer cells transfected with ATP6V1E1, pcDNA3.1
alone or control: A) ATP6V1E1 over-expression significantly decreased the ability
of MDA-MB231 breast cancer cells to survive and to form colonies as compared to
pcDNA3.1 alone and control (*P<0.05). Clonogenic assay demonstrates that long-
term survival of the cells is compromised after transfection with ATP6V1E1
constructs (n=3). B) A representative image of clonogenic assay plate after crystal
violet staining (n=3). ............................................... Error! Bookmark not defined.
Figure 4.25: ATP6V1E1 down regulation alter the cell cycle profile. A) Transfection with
ATP6V1E1 specific siRNAs (ATP6V1E1 siRNA2 and 4) caused a decrease in the
number of cells in G0/G1 accompanied with an increase in the number of cells in S
phase and G2/M phase 48 post-plating (n=3). *P<0.05 versus cells transfected with
(-) siRNA alone. ...................................................... Error! Bookmark not defined.
Figure 4.26: Transfection of MCF7 breast cancer cells with siRNAs2 and 4. After 28 h, the mean
number of CFUs was higher in ATP6V1E1-silenced MCF7 cells using both
siRNAs2 and 4 (n=3). ............................................. Error! Bookmark not defined.
Figure 4.27: Silencing of ATP6V1E1 in MCF7 breast cancer cells with two new ATP6V1E1
siRNA2 and 4: Cells were harvested at 72 h post-transfection, and re-plated for
assessment of cell viability after a further 48 h. A) Viable cell count was significantly
increased in cultures transfected with ATP6V1E1 siRNA4 followed by ATP6V1E1
siRNA2 as compared to controls (p<0.001). B) Total cell count was significantly
increased in cultures transfected with ATP6V1E1 siRNA4 followed by ATP6V1E1
siRNA2 compared to controls (p<0.001). Total and viable cell counts were measured
using hemocytometer and trypan blue (n=3). ......... Error! Bookmark not defined.
Figure 4.28: ATP6V1E1-silenced MCF7 cells re-plated for 72 h for assessment of cell viability.
A) Viable cell number was significantly increased in cultures transfected with
ATP6V1E1 siRNA4 followed ATP6V1E1 siRNA2 as compared to controls
(p<0.001). B) Total cell number was significantly increased in cultures transfected
with ATP6V1E1 siRNA4 followed by ATP6V1E1 siRNA2 compared to controls
(p<0.001). Total and viable cell counts were measured using FACS analysis (n=3).
Figure 4.29: Transfection of MCF7 breast cancer cells with two different siRNAs that target
different ATP6V1E1 or negative control siRNA. Cells were harvested after 72 h of
the transfection, and re-plated for assessment of cell survival after a further 48 h (A)
and 72 h (B), respectively. Total number of viable cells was significantly increased
in cultures transfected with ATP6V1E1 siRNA2 and siRNA4, in both cases. Total
and viable cell counts were measured using hemocytometer and trypan blue (n=3).
xiv
Figure 4.30: ATP6V1E1-silenced MCF7 cells re-plated for A) 48h and B) 72 h before cell viability.
A) percentage cell viability was significantly higher in cultures transfected with
ATP6V1E1 siRNA2 and 4 as compared to controls (p<0.001). B) The % viability
of cells re-plated for 72 h was similar in all ATP6V1E1-silenced MCF7 cells
including controls. Total and viable cell counts were measured using FACS analysis
and expressed as a percentage (n=3)....................... Error! Bookmark not defined.
Figure 4.31: The effect of ATP6V1E1 down-regulation on apoptosis rates MCF7 cells. MCF7
cells were silenced with ATP6V1E1 siRNA2 and 4, or negative siRNA or control
and apoptosis rates measured after 48h and 72 h post-plating using Muse®
MultiCaspase Assay Kit. A) ATP6V1E1-silenced cells exhibited significantly lower
apoptosis rates as compared to the controls (p<0.001); with ATP6V1E1 siRNA2
being a better silencer of the ATP6V1E1 gene than ATP6V1E1 siRNA4. B)
Similarly, the apoptosis rates after 72 h post-plating were significantly reduced in
ATP6V1E1 siRNA2 and 4 compared to controls (p<0.001); still with ATP6V1E1
siRNA2 showing better gene silencing effect (n=3).Error! Bookmark not
defined.
and apoptosis rates measured after 48h post-plating using A) Annexin or B) Acridine
orange. A) ATP6V1E1 downregulation in MCF7 breast cancer cells by ATP6V1E1
siRNA2 and 4 decreased the percentage of total apoptosis (p<0.001); B) ATP6V1E1
downregulation in MCF7 breast cancer cells by ATP6V1E1 siRNA2 and 4
decreased the percentage of total apoptosis (p<0.001). ATP6V1E1 siRNA4
exhibited a greater anti-apoptotic effect, thus a better gene silencing effect (n=3).
and apoptosis rates measured after 72 h post-plating using A) Annexin or B)
Acridine orange. ATP6V1E1 downregulation in MCF7 breast cancer cells by
ATP6V1E1 siRNA2 and 4 decreased the percentage of total apoptosis in both cases
(p<0.001) (n=3)....................................................... Error! Bookmark not defined.
Figure 4.34: Effects of ATP6V1E1 silencing on the expression of endogenous ATP6V1E1 in

MCF7 cells. Silencing reduced the relative expression of ATP6V1E1 in MCF7 cells
(n=3). ...................................................................... Error! Bookmark not defined.
Figure 4.35: Effects of ATP6V1E1 silencing on the expression of endogenous ATP6V1E1 in

MDA-MB231 cells. Silencing reduced the relative expression of ATP6V1E1 in
MCF7 cells.............................................................. Error! Bookmark not defined.
Figure 4.36: The effect of ATP6V1E1 down-regulation on apoptosis rates in MDA-MB231 breast
cancer. MDA-MB231 cells were silenced with ATP6V1E1 siRNA1 and 4, or (-
)siRNA or control and apoptosis rates measured after 48 h and 72 h post-plating
using Muse® MultiCaspase Assay Kit. A) ATP6V1E1-silenced cells exhibited
significantly lower apoptosis rates as compared to the controls at 48 h (p<0.001). B)
Similarly, the apoptosis rates at 72 h post-plating were significantly reduced in
ATP6V1E1 siRNA2 and 4 compared to controls (n=3). (p<0.001). .............. Error!
Figure 4.37: A)After 48 h post-plating ATP6V1E1 down-regulation in MDA-MB-231 breast

cancer cells by siRNA1 and 4 decreased the % apoptosis as evaluated using
Annexin; B. after 72 h ATP6V1E1 downregulation in MDA-MB-231 breast cancer
cells by ATP6V1E1 siRNA1 and 4 decreased % apoptosis as evaluated using
Annexin (n=3). ........................................................ Error! Bookmark not defined.
xv
Figure 4.38: A) After 48 h post-plating ATP6V1E1 down-regulation in MDA-MB-231 breast
cancer cells by siRNA1 and 4 decreased the % apoptosis as evaluated using
Acridine orange test; B. after 72 h ATP6V1E1 downregulation in MDA-MB-231
breast cancer cells by ATP6V1E1 siRNA1 and 4 decreased % apoptosis as
evaluated using Acridine orange (n=3). .................. Error! Bookmark not defined.
Figure 4.39: ATP6V1E1 down-regulation in MDA-MB231 breast cancer cells after A) 48h and B)
72 h post-plating. After 48 h post-plating there was an increase in the total number
of cells transfected with siRNA1 and 4 as compared to controls (P<0.01). Similar
results were observed in cells transfected with siRNA1 and 4(n=3) (P<0.01).
Figure 4.40: A) ATP6V1E1-silenced MDA-MB231 cells using siRNA1 & 4 and then re-plated for
48 h and assessed for cell viability using hemocytometer exhibited higher viable cell
count compared to the controls (P<0.05). B) similar results were observed when the
cells were analyzed by Muse (P<0.05). Total and viable cell counts were measured
using FACS analysis (n=3). .................................... Error! Bookmark not defined.
Figure 4.41: A) ATP6V1E1-silenced MDA-MB231 cells using siRNA1 & 4 and then re-plated for
72 h and assessed for cell viability using hemocytometer exhibited higher viable cell
count compared to the controls (P<0.05). B) similar results were observed when the
cells were analyzed by Muse (P<0.05). Total and viable cell counts were measured
using FACS analysis (n=3). .................................... Error! Bookmark not defined.
Figure 4.42: ATP6V1E1-silenced MCF7 cells re-plated for A) 48h and B) 72 h before cell viability.
A) percentage cell viability was significantly higher in cultures transfected with
ATP6V1E1 siRNA2 and 4 as compared to controls (p<0.001). B) The % viability
of cells re-plated for 72 h was similar in all ATP6V1E1-silenced MCF7 cells
including controls. Total and viable cell counts were measured using FACS analysis
and expressed as a percentage (n=3). ...................... Error! Bookmark not defined.
Figure 4.43: Colony formation in long-term clonogenic assays after the transfection of the MDA-
MB231 breast cancer cells with the ATP6V1E1 specific siRNAs 1& 4. The siRNAs
increased the ability of these cells to survive and form colonies as compared to the
control cells transfected with (-) siRNA (n=3) (*P<0.05).Error! Bookmark not
defined.
Figure 4.44: ATP6V1E1 siRNA transfection of MDA-MB231 breast cancer cells with ATP6V1E1
siRNA1 & 4. All siRNAs caused a decrease in the percentage of cells in sub-G0
phase. siRNAs appear to protect against cell death as shown by the decrease in the
cells in sub-G0 phase (n=3) compared to the controls (n=3) (*P<0.05). ........ Error!
Figure 4.45: TEM images showing the cell ultra-structure of MCF7 cells transfected with
pcDNA3.1. Actin filaments (Yellow arrows), vacuoles (red arrows) nucleus and
nucleolus (green arrows). Scale bars = 5 μm. ......... Error! Bookmark not defined.
Figure 4.46: TEM images showing the cell ultra-structure of MCF7 cells transfected with
ATP6V1E1. A-F replicates of cell transfected with ATP6V1E1. Organelles and
intracellular structures can be seen including nucleus and nucleolus (yellow arrow),
vacuoles (blue arrows), lysosomes (green arrows) actin filaments (black arrows).
Noticeably, ATP6V1E1 resulted in the induction of vacuoles (red arrows)
characterized by an increase in both the number and size of the vacuoles. The grey
bodies (red arrows), look like lipid droplets which were also seen in the control.
Cytoplasmic density and disruption are visible suggesting apoptotic. Scale Scale
bars = 5, 10 μm. ...................................................... Error! Bookmark not defined.
xvi
Figure 4.47: TEM images showing the cell ultra-structure of MDA-MB-231 cells transfected with
pcDNA3.1. The pcDNA3.1 did not seem to affect the cell ultra-structure. Several
intra-cellular structures can be seen including nucleus and nucleolus (green arrows),
mitochondria (blue arrows), microtubules (red arrows) and lysosomes (black
arrows). Vacuoles were absent. Scale bars = 5, 10 µm.Error! Bookmark not
defined.
Figure 4.48: TEM images showing the cell ultra-structure of MDA-MB-231 cells transfected with
ATP6V1E1. Several intra-cellular structures can be seen including nucleus and
nucleolus (green arrows). Viral-like structures can be seen in the nucleus (red
arrows). Vacuoles are absent. Scale bars =5 µm, 5 µm, 10 µm, 0.5µm, for A, B, C,
and D, respectively. ................................................ Error! Bookmark not defined.
Figure 5.1: Effect of ATP6V1E1 over-expression on the expression of actin protein in MCF7. By
visual inspection, normal expression of ATP6V1E1 (control and pcDNA3.1)
exhibited a higher actin content compared to ATP6V1E1-overexpressing cells.
Thus, ATP6V1E1 overexpression resulted in the reduction of actin labelling Scale
bars= 200 μm. ......................................................... Error! Bookmark not defined.
Figure 5.2: Effect of ATP6V1E1 over-expression on the expression of actin protein in MDA-MB-
231 cells. The intensity of actin protein in MDA-MB-231 cells was higher in control
and negative control (pcDNA3.1) compared to ATP6V1E1-overexpressing cells
(pcDNA3.1-ATP6V1E1). Scale bars= 200 μm. ..... Error! Bookmark not defined.
Figure 5.3: The profile graphs represent the fluorescence intensity across the lines drawn on each
image. The expression level of the actin protein was higher in the control and
negative control (pcDNA3.1) MCF7 cells (A and B) than ATP6V1E1-
overexpressing cells, C (pcDNA3.1-ATP6V1E1). This was evidenced by a higher
peak amplitude in the profile graphs. Scale bar=200 μm.Error! Bookmark not
defined.
Figure 5.4: The profile graphs represent the fluorescence intensity across the lines drawn on each
image. The expression level of the actin protein was higher in the control and
negative control (pcDNA3.1) MDA-MB-231 cells (A and B) than ATP6V1E1-
overexpressing cells, C (pcDNA3.1-ATP6V1E1). This was evidenced by a higher
peak amplitude in the profile graphs. Scale bar= 200 μm.Error! Bookmark not
defined.
Figure 5.5: Actin protein expression in MCF7 and MDA-MB-231 cells: A) In MCF7 cells,
ATP6V1E-treated cells exhibited a significantly lower actin protein expression level
(count per pixel) (~11) as compared to both untreated control and negative
PcDNA3.1 control (~29, 32), (n=10) (P < 0.05); B) In MDA-MB-231 cells,
ATP6V1E1-transfected cells exhibited actin levels of ~5 as compared to controls
(9, 10), (n=10) (p<0.05); In general, the expression of actin protein was higher in
MCF7 than in MDA-MB-231 cells......................... Error! Bookmark not defined.
Figure 5.6: The comparative actin expression in MCF7 and MDA-MB-231 cells; A) In MCF7 cells,
the intensities were ~10, 34 and 36, for PcDNA3. 1-ATP6V1E1-treated, PcDNA3.
1-treated and untreated control cells, respectively. The difference was statistically
significant (n=10) (p<0.0001); B) In MDA-MB-231 cells, the intensities were ~6,
13 and 12, for PcDNA3. 1-ATP6V1E1-treated, PcDNA3. 1-treated and untreated
control cells, respectively. The difference was statistically significant (n=10)
(p<0.0001). The intensities in MCF7 cells were generally higher compared to MDA-
MB-231 cells........................................................... Error! Bookmark not defined.
Figure 5.7: Negative control cells in both MCF7 cells (A-C) and MDA-MB-231 cells (D-E) showed
clear evidence of cell ruffles containing tubulin and a polarized distribution that
xvii
reflected an active normal cytoskeleton with visible microtubular filaments.
pcDAN3.1-ATP6V1E1 transfected cells showed round morphology, lacking
microtubular filaments. Scale bars= 200 μm. ......... Error! Bookmark not defined.
Figure 5.8: pcDNA3.1 cells showed some evidence of cell ruffles containing actin and a polarized
distribution that reflected an active normal cytoskeleton with visible actin fibers.
pcDAN3.1-ATP6V1E1 transfected cells showed round morphology, lacking actin
filaments. Scale bars= 200 μm. ............................... Error! Bookmark not defined.
Figure 5.9: Tubulin protein expression in MCF7 and MDA-MB-231 cells: A) In MCF7 cells,
pcDNA3.1, and ATP6V1E-treated cells exhibited tubulin expression levels of ~80,
and ~20, respectively (n=10) (P<0.0001); B) In MDA-MB-231 cells, PcDNA3.1
and ATP6V1E1-transfected cells exhibited tubulin expression levels of ~21; ~23;
and ~13, respectively (n=10). ................................. Error! Bookmark not defined.
Figure 5.10: The profile graphs represent the expression intensity of tubulin across the lines drawn
on each image. The expression level of the tubulin protein was lower in the negative
control (pcDNA3.1) MDA-MB-231 cells than in ATP6V1E1-overexpressing cells
(pcDNA3.1-ATP6V1E1), evidenced by a greater peak amplitude in the profile
graph. Scale bars= 100 μm...................................... Error! Bookmark not defined.
Figure 5.11: The profile graphs represent the expression intensity of tubulin across the lines drawn
on each image. The expression level of the tubulin protein was higher in the negative
control (pcDNA3.1) MDA-MB-231 cells than in ATP6V1E1-overexpressing cells
(pcDNA3.1-ATP6V1E1), evidenced by a greater peak amplitude in the profile
graph. Scale bars = 100 μm..................................... Error! Bookmark not defined.
Figure 5.12: Tubulin protein intensities in MCF7 and MDA-MB-231 cells: A) In MCF7 cells
negative (PcDNA3.1) control cells exhibited a significantly higher tubulin
intensities compared to ATP6V1E1-treated cells (~85 vs. ~35). The intensity
difference was statistically significant (n=10) (P<0.0001); B). In MDA-MB-231
cells, PcDNA3.1-treated cells exhibited a higher tubulin intensity compared to
ATP6V1E1-treated cells (~112 Vs. ~50) (n=10) (P<0.0001).Error! Bookmark
not defined.
Figure 5.13: Scratch wound assay showing the effect of ATP6V1E1 down-expression on migration
of MCF7 cancer cells (ATP6V1E1-siRNA2, 4) compared to controls (negative
siRNA control and control). The degree of cell migration appears to increase with
time from 0 to 96 h. Difference in the migration of ATP6V1E1-siRNA2, 4 and
controls was analyzed using Image J software. ...... Error! Bookmark not defined.
Figure 5.14: Scratch wound assay showing the effect of ATP6V1E1 down-expression on migration
of MDA-MB-231 cancer cells (ATP6V1E1-siRNA2, 4) compared to controls
(negative siRNA control and control). The degree of cell migration appears to
increase with time from 0 to 96 h. Difference in the migration of ATP6V1E1-
siRNA1, 4 and controls was analyzed using Image J software.Error! Bookmark
not defined.
Figure 5.15: Wound scratch assay of MCF7 cancer cells showing the effect of ATP6V1E1
downregulation on cell migration. Clearly, ATP6V1E1 downregulation generally
resulted in higher % of wound closure than controls, with ATP6V1E1-siRNA2,
exhibiting a higher rate than ATP6V1E1-siRNA4 (n=4).Error! Bookmark not
defined.
Figure 5.16: Wound scratch assay of MDA-MB-231 cancer cells showing the effect of ATP6V1E1
downregulation on cell migration. ATP6V1E1 downregulation generally resulted
xviii
in higher % of wound closure than controls, with ATP6V1E1-siRNA1, exhibiting a
higher rate than ATP6V1E1-siRNA4 (n=4). .......... Error! Bookmark not defined.
Figure 6.1: MCF7 stained with the pH-sensitive LysoSensor™ Green DND-189 dye. By visual
inspection, ATP6V1E1-overexpressimg cells exhibited a greater labelling intensity
compared to controls (untreated controls and negative pcDNA3.1 control). The same
cells are shown in the DIC microscopy images (lower panels). ATP6V1E1-
overexpressimg cells exhibited a greater acidity due to increased acidity in
intracellular organelles/structures. In general, the LysoSensor-labelling was
peripheral with little cytoplasmic labelling. Scale bars= 150 μm.Error! Bookmark
not defined.
Figure 6.2: MDA-MB-231 cells stained with the pH-sensitive LysoSensor™ Green DND-189 dye.
By visual inspection, ATP6V1E1-overexpressimg cells exhibited a greater labelling
intensity compared to controls. ATP6V1E1-overexpressimg cells exhibited a greater
acidity due to increased acidity in intracellular organelles/structures. In general, the
LysoSensor-labelling was predominantly cytoplasmic with little peripheral
labelling. Scale bar= 150 μm. ................................. Error! Bookmark not defined.
Figure 6.3: The intensity of LysoSensor™-labelled cells: A) ATP6V1E1-overexpressing MCF7

cells exhibited a significantly greater intensity compared to controls (n=10)
(p<0.01). The negative pcDNA3.1 control exhibited a greater intensity than the
untreated control; B) In MDA-MB-231 cells, the ATP6V1E1-overexpression
resulted in a significantly higher intensity compared to controls (n=10) (p<0.0001).
Figure 6.4: MCF7 stained with the pH-sensitive LysoSensor™ Green DND-189 dye; By visual
inspection, downregulation of ATP6V1E1 protein using siRNA2 and siRNA4
resulted in a diminished intensity, as compared to untreated control and negative
siRNA control. Loss of ATP6V1E1 protein resulted in reduced acidity in in
intracellular organelles/structures, leading to a reduced intensity. Scale bars = 200
Figure 6.5: MDA-MB-231 cells stained with the pH-sensitive LysoSensor™ Green DND-189 dye;
By visual inspection, reduced or loss of ATP6V1E1 protein via siRNA2 and siRNA4
knockdown, resulted in a diminished intensity, as compared to untreated control and
negative siRNA control. Loss of ATP6V1E1 protein resulted in reduced acidity in
in intracellular organelles/structures, leading to a reduced intensity. Scale bars= 200
Figure 6.6: The intensity of LysoSensor™-labelled cells: A) ATP6V1E1-siRNA2 and 4 MCF7

cells exhibited a significantly lower intensity compared to untreated control and
negative siRNA control (n=10) (p<0.05). siRNA2 exhibited a greater intensity than
siRNA4, while untreated control was higher than negative siRNA control; B) In
MDA-MB-231 cells, the ATP6V1E1-siRNA1 and 4 cells exhibited a significantly
lower intensity compared to controls (n=10) (p<0.0001). The intensity in siRNA1
cells was higher than siRNA4. Thus, loss of ATP6V1E1 resulted in reduced acidity
in MDA-MB-231 cells. ........................................... Error! Bookmark not defined.
Figure 6.7: Light microscopy images showing time-dependent (2-24 h) survival of MCF7 cells
grown under acidic condition (pH 6.5): Cells grown in the acidic pH exhibited
increased time-dependent survival from 2 h to 24 h post-plating. This survival trend
appeared to be similar to controls though acidity exhibited a higher survival than
controls.................................................................... Error! Bookmark not defined.
Figure 6.8: Light microscopy images showing time-dependent (2-24 h) survival of MDA-MB-231
cells grown under acidic condition (pH 6.5): Cells grown in the acidic pH exhibited
xix
increased time-dependent survival from 2 h to 24 h post-plating. This survival trend
appeared quite similar to controls though acidity exhibited a higher survival than
controls.................................................................... Error! Bookmark not defined.
Figure 6.9: Scale bar graphs of time-dependent (2-24 h post-plating) viable cell count for MCF7
cells grown under acidic condition (pH 6.5): Acidic and control cells exhibited
increased viable cell count from 2 h to 24 h post-plating. After 24 h, acidic cells
exhibited a significantly higher viable cell count than controls (p<0.001). .... Error!
Figure 6.10: Scale bar graphs of time-dependent (2-24 h post-plating) viable cell count for MDA-
MB-231 cells grown under acidic condition (pH 6.5) compared to controls: Acidic
and control cells exhibited increased viable cell count from 2 h to 24 h post plaiting.
Acidic and control cells exhibited increased viable cell count from 2 h to 24 h post-
plating. After 24 h, acidic cells exhibited a significantly higher viable cell count than
controls (p<0.001)................................................... Error! Bookmark not defined.
Figure 6.11: Light microscopy images showing time-dependent (2-24 h) survival of MCF7 cells
grown under alkaline condition (pH 8.5): Cells grown in the alkaline pH exhibited
decreased time-dependent survival from 2 h to 24 h post-plating, while the controls
exhibited increased survival trend. ......................... Error! Bookmark not defined.
Figure 6.12: Light microscopy images showing time-dependent (2-24 h) survival of MDA-MB-
231 cells grown under alkaline condition (pH 8.5): Cells grown in the alkaline
conditions exhibited decreased while controls exhibited increased time-dependent
survival from 2 h to 24 h post-plating. .................... Error! Bookmark not defined.
Figure 6.13: Scale bar graphs of time-dependent (2-24 h post-plating) viable cell count for MCF7
cells grown under alkaline condition (pH 8.5): Alkaline cells exhibited decreased
while controls exhibited increased viable cell count from 2 h to 24 h post-plating.
Alkaline cells exhibited significantly lower viable cell count than controls after 6 h,
14 h and 24 h (p<0.0001). ....................................... Error! Bookmark not defined.
Figure 6.14: Scale bar graphs of time-dependent (2-24 h post-plating) viable cell count for MDA-
MB-231 cells grown under alkaline condition (pH 8.5): Alkaline cells exhibited
decreased while controls exhibited increased viable cell count from 2 h to 24 h post-
plating. Alkaline cells exhibited significantly lower viable cell count than controls
after 6 h (p<0.001), 14 h (p<0.0001) and 24 h, (p<0.0001).Error! Bookmark not
defined.
Figure 6.15: Effect of ATP6V1E1-overexpression on the extracellular acidity of MCF7 cells. A)

After 48 h post-plating ATP6V1E1-overexpression cells exhibited higher pH
compared to untreated and negative pcDNA3.1 control; B) After 72 h post-plating,
pH increased in controls but remained constant at ~8 in ATP6V1E1-overexpression
cells. Loss of ATP6V1E1 resulted in diminished acidity.Error! Bookmark not
defined.
Figure 6.16: Effect of ATP6V1E1-downregulation on the extracellular acidity of MCF7 cells. A)

After 48 h post-plating ATP6V1E1-downregulated cells (siRNA2, 4) exhibited
lower pH compared to untreated and negative siRNA control; B) After 72 h post-
plating, acidity increased to a pH of 8.2 in controls and ~8 in siRNA2 and 4 cells.
Loss of ATP6V1E1 resulted in diminished acidity. Error! Bookmark not defined.
Figure 6.17: Effect of ATP6V1E1-overexpression on the extracellular acidity of MDA-MB-231

cells. A) After 48 h post-plating ATP6V1E1-overexpression cells exhibited higher
pH (~8) compared to untreated and negative pcDNA3.1 control (~7.5); B) After 72
h post-plating, pH increased in controls as well as in ATP6V1E1-overexpressing
xx
cells, with all cells exhibiting a pH of ~8.5. Increased expression of ATP6V1E1
resulted in increased acidity. ................................... Error! Bookmark not defined.
Figure 6.18: Effect of ATP6V1E1-downregulation on the extracellular acidity of MDA-MB-231

cells. A) After 48 h post-plating ATP6V1E1-downregulated cells (siRNA1, 4)
exhibited lower pH (~6.9) compared to untreated and the negative siRNA control
(~7.2); B) After 72 h post-plating, acidity increased with controls exhibiting
significantly higher pH (~ 7.9) than siRNA1 and 4 cells (~7.4, ~7.7; p<0.0001,
<0.001). Loss of ATP6V1E1 resulted in diminished acidity.Error! Bookmark not
defined.
Figure 6.19: Effect of RPMI medium acidification on the intensity of tubulin in MCF7 cancer cells:
Cells cultured in acidified medium exhibited a higher (brighter) tubulin intensity as
compared to control throughout the time intervals (2, 4, 6 and 14 h). Scale bars =
100 μm. ................................................................... Error! Bookmark not defined.
Figure 6.20: Effect of RPMI medium acidification on the intensity of tubulin in MDA-MB-231
cancer cells: Cells cultured in acidified medium exhibited a higher (brighter) tubulin
intensity as compared to control throughout the time intervals and it diminished in
pH 6.5 over the time period (2, 4, 6 and 14 h). Scale bars= 100 μm. ............. Error!
Figure 6.21: Quantitative analysis of 40 cell samples for tubulin intensity: A) MCF7 cancer cells
grown on acidified RPMI medium (6.5) exhibited a higher tubulin intensity than
control (~80 vs. ~65); B) In MDA-MB-231 cells, the intensity was higher in
acidified medium compared to controls (~100 vs. 60). The difference was
statistically significant in both cases (p<0.0001). ... Error! Bookmark not defined.
xxi
List of abbreviations
7-AAD 7-Amino-Actinomycin D
ACSS2 Acyl-CoA Synthetase Short-chain family member 2
AIF Apoptosis Inducing Factor
APAF1 Apoptotic protease-activating factor 1
ASIC Acid-Sensing Ion Channels
ATCC American Type Culture Collection
ATP Adenosine tri-phosphate
B.C Before Christ
Bcl-2 B-cell lymphoma 2
BMPs Bone morphogenetic proteins
BRCA1 Breast Cancer Associated Gene 1
BRCA2 Breast Cancer Associated Gene 2
BSA Bovine Serum Albumin
CA Carbonic Anhydrase Enzymes
CA9 Carbonic Anhydrase 9
cAMP Cyclic Adenosine Monophosphate
Caspase Cysteine-aspartic proteases
CFUs Colony- Forming Units
CMV Cytomegalovirus
DCIS Ductal Carcinoma in Situ
DIC Differential Interference Contrast
DISC Death Inducing Signaling Complex
DMSO Dimethyl Sulfoxide
DNA Deoxyribonucleic Acid
ECL Enhanced Chemiluminescence
ECM Extracellular matrix
EDTA Ethylenediaminetetraacetic Acid
EMT Epithelial–Mesenchymal Transition
ER Estrogen Receptor
ETOH Ethanol
FACS Fluorescence Activated Cell Sorting
FADD Fas-Associated Death Domain Protein
Fas First Apoptosis Signal
Fasl Fas Ligand
FBS Foetal Bovine Serum
GBM Glioblastoma multiforme
GFP Green Fluorescent Protein
GPCR G-protein-Coupled Receptors
GPR4 G-protein coupled receptor 4
GS Goat Serum
HEK293 Human Embryonic Kidney 293
Her-2/neu Human epidermal growth factor receptor 2
HIF1α hypoxia-inducible factor-1alpa (HIF1α)
HIV Human Immunodeficiency Virus
HR+ Hormone Receptor-positive
LAMP-1 Lysosomal-Associated Membrane Protein 1
LKB1 Liver kinase B1
22
MAPK Mitogen-Activated Protein Kinase
MCF7 Michigan Cancer Foundation 7 Breast Cancer Cell Line
MCT Multiple Comparison Test
MCTs Monocarboxylate Transporters
MDA-MB-231 Breast cancer cell line derived from metastatic site
MFI(+) Positive Mean Fluorescence Intensity
MMP Matrix Metalloproteinase
MMP-9 Matrix metallopeptidase 9
mRNA Messenger Ribonucleic Acid
N-cadherin Neural cadherin
NGF Nerve growth factor
NHE1 Sodium–hydrogen antiporter 1
NHEi Na+/H+ transporters/exchangers 1
OGR1 Ovarian cancer G-protein-coupled receptor
p53 Tumor Protein 53
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate Buffered Saline
PfnI Profilin I
PJS Puetz-Jeghers syndrome
PP Proton Pumps
PR Progesterone Receptor
PS Phosphatidylserine
PTEN Phosphatase and Tensin Homolog
PVDF Polyvinylidene Difluoride
RNA Ribonucleic Acid
RPMI Roswell Park Memorial Institute (culture medium)
SDS Sodium Dodecyl Sulphate
SEM Standard Error of the Mean
siRNA Small Interfering RNA
SKBR3 breast cancer cell line isolated by the Memorial Sloan–Kettering Cancer
Center
Smac Second mitochondrial protein
SREBP2 Sterol Regulatory Element-Binding Protein 2
STK11 Serine/threonine kinase 11
TBAs Tubulin-Binding Agents
TBS Tris Buffered Saline
TBST Tris Buffered Saline, 0.1% TWEEN® 20
TDAG8 T cell death-associated gene 8
TEM Transmission Electron Microscopy
TGF Transforming Growth Factor
TNBC Triple-Negative Breast Cancer
TNF Tumor Necrosis Factor
TP Thymidine phosphorylate
TP Thymidine Phosphorylate
TRPV1 Transport Receptor Potential Channel Vanilloid Subfamily 1
TSP-1 Thrombospondin 1
V-ATPase Vacuolar-type H + -ATPase
VEGF Vascular Endothelial Growth Factor
VEGF-A Vascular endothelial growth factor A
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Background and aims: The aggressiveness of breast cancer is influenced by multiple

Figure 1.12: Construction of a pH-sensor device. G-protein-coupled with receptor (TDAG8)

Figure 3.2: Immunocytochemistry analysis of ATP6V1E1 protein in the PFA-fixed MDA-MB-231

Figure 3.3: Immunocytochemical analysis of the ATP6V1E1 protein in PTA-PFA-fixed MCF7

Figure 4.1: ATP6V1E1 over-expression in MCF7 was achieved using pcDNA3.1-V-ATP6V1E1

Figure 4.8: Effects of ATP6V1E1 transfection on the expression of endogenous ATP6V1E1 in

Figure 4.16: Effects of ATP6V1E1 transfection in MDA-MB231 cells. A) Immunoblot of

Figure 4.34: Effects of ATP6V1E1 silencing on the expression of endogenous ATP6V1E1 in

Figure 4.35: Effects of ATP6V1E1 silencing on the expression of endogenous ATP6V1E1 in

Figure 4.37: A)After 48 h post-plating ATP6V1E1 down-regulation in MDA-MB-231 breast

Figure 6.3: The intensity of LysoSensor™-labelled cells: A) ATP6V1E1-overexpressing MCF7

Figure 6.6: The intensity of LysoSensor™-labelled cells: A) ATP6V1E1-siRNA2 and 4 MCF7

Figure 6.15: Effect of ATP6V1E1-overexpression on the extracellular acidity of MCF7 cells. A)

Figure 6.16: Effect of ATP6V1E1-downregulation on the extracellular acidity of MCF7 cells. A)

Figure 6.17: Effect of ATP6V1E1-overexpression on the extracellular acidity of MDA-MB-231

Figure 6.18: Effect of ATP6V1E1-downregulation on the extracellular acidity of MDA-MB-231

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