The Pennsylvania State University

The Graduate School

Department of Chemical Engineering

EVOLUTION OF CELL GENERATED FORCES DURING EPITHELIAL

MYOFIBROBLAST TRANSITION

A Thesis in

Chemical Engineering

by

Sandeep Mouli Nalluri

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Master of Science

August 2015

The thesis of Sandeep Mouli Nalluri was reviewed and approved* by the following:

Esther W Gomez

Assistant Professor of Chemical Engineering &

Assistant Professor of Bioengineering

Thesis Advisor

Manish Kumar

Assistant Professor of Chemical Engineering

Phillip E. Savage

Head of the Department of Chemical Engineering

Walter L. Robb Family Endowed Chair

*Signatures are on file in the Graduate School

iii

ABSTRACT

Myofibroblasts are cells that aid in wound healing and upon aberrant activation they can promote

diseases including organ fibrosis and cancer. Myofibroblasts can develop from epithelial cells

through an epithelial-mesenchymal transition (EMT), which is characterized by loss of cell-cell

junctions, changes in gene expression and increased cell motility. While many transcriptional

programs that govern EMT are well studied, little is known about how the cytoskeleton

contributes to EMT progression. Here, we find that the expression levels of cytoskeletal

associated proteins are increased during transforming growth factor (TGF)-beta 1 induced EMT

which correlates with cytoskeletal rearrangements and increased exertion of contractile forces by

cells. Furthermore, we find that inhibition of ERK 1/2 and p38 MAPK dramatically impacts the

acquisition of myofibroblast characteristics during EMT. These studies identify key cytoskeletal

associated signaling pathways that regulate the development of myofibroblasts and suggest

approaches to promote myofibroblast development for reparative functions or to abrogate the

activation of myofibroblasts in pathological contexts. Furthermore the regulation of focal

adhesion proteins during EMT in cancer cells with extracellular matrix stiffness was discussed.

iv

TABLE OF CONTENTS

List of Figures .......................................................................................................................... v

List of Tables ........................................................................................................................... vi

Acknowledgements .................................................................................................................. vii

Chapter 1 Background ............................................................................................................. 1

Epithelial-mesenchymal transition ........................................................................... 1 Evolution of cell generated forces and cytoskeletal signaling during EMT............. 3 Role of ERK and p38 MAPK in development of myofibroblasts through EMT ..... 4

Chapter 2 Evolution of cell generated forces ........................................................................... 5

Introduction .............................................................................................................. 5 Materials and Methods ............................................................................................. 5 Results ...................................................................................................................... 10 Discussion ................................................................................................................ 15

Chapter 3 Role of ERK ½ and p38 MAPK in acquisition of myofibroblast characteristics .... 18

Introduction .............................................................................................................. 18 Results ...................................................................................................................... 18 Discussion ................................................................................................................ 28

Chapter 4 Regulation of focal adhesion proteins expression during epithelial

mesenchymal transition of HCC1954 cells with matrix stiffness .................................... 31

Introduction .............................................................................................................. 31 Materials and Methods ............................................................................................. 32 Results ...................................................................................................................... 33 Conclusion ................................................................................................................ 35

Chapter 5 Conclusion and Future ............................................................................................ 37

References ........................................................................................................................ 39

v

LIST OF FIGURES

Figure 1-1 Epithelial mesenchymal transition is characterized by loss of cell-cell

junctions, acquisition of an elongated morphology and formation of stress fibers.

Mesenchymal cells further progress through EMT to form myofibroblasts. ................... 2

Figure 2-1 TGFβ1induces changes in epithelial and mesenchymal proteins. NMuMG

cells treated with control and TGFβ1 are stained for E-cadherin, α-SMA,

tropomyosin, and caldesmon at 24, 48 and 72 h. Scale bar: 25 μm. ................................ 11

Figure 2-2 Western blot of control and TGFβ1 treated cells at 24, 48 and 72 h for E-

cadherin, α-SMA, tropomyosin, p-caldesmon, caldesmon, and α-tubulin, the loading

control. The expression of actin binding proteins caldesmon, p-caldesmon and

tropomyosin is upregulated during TGFβ1 induced EMT and α-SMA is expressed at

72 h following the treatment with TGFβ1. ....................................................................... 12

Figure 2-3 During the progression of EMT the actin cytoskeleton rearranges from a

cortical architecture to prominent stress fibers. Focal adhesion number and area

increase during the progression of EMT. Scale bar: 25 μm. ............................................ 13

Figure 2-4 Cells exhibit increased focal adhesions following induction of EMT. (A)

Immunofluorescence staining for vinculin. Scale bar: 25 μm. Quantification of (B)

focal adhesion number and (C) focal adhesion area. ....................................................... 14

Figure 2-5 TFM analysis of NMuMG cells (A) Maps of traction stresses along the cells.

Average (B) area, ##p<0.001 and #p<0.0001, *p<0.001 in comparison to 24 h

TGFβ1 (C) force, # p < 0.0001; *** p < 0.0001 in comparison to 24 h and 48 h

TGFβ1 (D) net contractile moment # p < 0.000001; *** p < 0.01 in comparison to

24 h and 48 h TGFβ1 and (E) traction stress #p<0.5, ##p<0.0001 and *p<0.01 in

comparison to all other samples. ...................................................................................... 16

Figure 2-6 TFM analysis of MDCK cells treated with control or TGFβ1 at 72 h. (A)

Maps of traction stresses along the cells. Average (B) area (C) force (D) net

contractile moment and (E) traction stress. *p<0.0001 in comparison to respective

control samples. ............................................................................................................... 17

Figure 3-1 Western blot showing (A) ERK1/2 phosphorylation at 24, 48 and 72 h in

TGFβ1 and control treated NMuMG cells (B) ERK phosphorylation upon treatment

with TGFβ1 + U0126 and TGFβ1+dmso at 72 h (C) ERK phosphorylation,

expression of α-SMA, tropomyosin, caldesmon and p-caldesmon at 72 h in NMuMG

cells treated with control/TGFβ1 plus DMSO/U0126, also shown are α-SMA,

tropomyosin, caldesmon and p-caldesmon. ..................................................................... 19

Figure 3-2 Real time - PCR at 72 h in NMuMG cells treated with control or TGFβ1 plus

DMSO or U0126. Transcript levels for (A) E-cadherin, **p<0.001, #p<0.05, (B) α-

SMA, *p<0.01, #p<0.5 compared to TGFβ1 plus DMSO, (C) caldesmon, #p<0.5,

*p<0.01 compared to control plus DMSO, and (D) tropomyosin, *p<0.01, #p<0.5

compared to control plus DMSO. .................................................................................... 20

vi

Figure 3-3 Staining for actin at 72 h in NMuMG cells treated with control or TGFβ1 plus

DMSO or U0126. U0126 did not block TGFβ1 induced stress fibers formation at 72

h. Scale bar ~ 25μm. ........................................................................................................ 21

Figure 3-4 U0126 did not block TGFβ1 induced focal adhesions formation at 72 h. (A)

Immunofluorescence staining for vinculin at 72 h in NMuMG cells treated with

control or TGFβ1 plus DMSO or U0126, and quantification of (B) focal adhesion

area and (C) focal adhesion number. ................................................................................ 22

Figure 3-5 Traction force microscopy at 72 h post the treatment of NMuMG cells treated

with control/TGFβ1 plus DMSO/U0126 was used to generate (A) traction stress

maps, and to calculate (B) cell area, #p<0.0001, *p<0.0001 comparison to TGFβ1 +

DMSO, (C) force exerted by the cells, ##p<0.001, #p<0.0001, *p<0.0001

comparison to TGFβ1 + DMSO, (D) net contractile moment of the cells, #p<0.0001,

*p<0.0001 comparison to TGFβ1 + DMSO, and (E) average traction stress exerted

by the cells, p<0.05 in comparison to control plus DMSO. N=50 cells per condition

and ##p<0.001, #p<0.0001, *p<0.0001 comparison to TGFβ1 + DMSO. ...................... 23

Figure 3-6 Western blot showing expression of p-ERK1/2, ERK1/2, α-SMA, E-cadherin,

and α-tubulin, the loading control, in MDCK cells at 72 h treated with control or

TGFβ1 plus DMSO or U0126. ......................................................................................... 25

Figure 3-7 Traction force microscopy at 72 h post the treatment of NMuMG cells treated

with control or TGFβ1 plus DMSO or SB203580. (A) cell area, (B) force exerted

by the cells, (C) net-contractile moment of the cells, and (D) average traction stress

exerted by the cells; #p<0.05 and *p<0.0001. ................................................................. 26

Figure 3-8 Real time - PCR at 72 h in NMuMG cells treated with control or TGFβ1 plus

DMSO or SB203580. Transcript levels for (A) E-cadherin, *p<0.001, (B) α-SMA,

(C) caldesmon, and (D) tropomyosin. .............................................................................. 27

Figure 3-9 Traction force microscopy at 72 h on MDCK cells treated with control or

TGFβ1 plus DMSO or U0126. (A) traction maps showing traction stress across the

cells, (B) cell Area, #p<0.001 and *p<0.001 compared to TGFβ1 plus DMSO, (C)

forces exerted by the cells, #p<0.001 and *p<0.001 compared to TGFβ1 plus

DMSO, (D) net contractile moment of the cells, #p<0.001 and ##p<0.05, and (E)

average traction stress exerted by the cells, #p<0.001 and ##p<0.05. Minimum of 50

cells were used per condition over three different experiments. ...................................... 29

Figure 4-1 Western blot showing expression of vinculin, LPP, and GAPDH, the loading

control, in HCC1954 cells at 96 h treated with control or TGFβ1 in (A) low serum

medium and (B) high serum medium. .............................................................................. 34

Figure 4-2 Immunofluorescence staining for vinculin in HCC1954 cells at 96 h post the

treatment of cells with control or TGFβ1 on 130 Pa, 1800 Pa and 6000 Pa gels. ............ 34

Figure 4-3 Traction force microscopy analysis of HCC1954 cells treated with control or

TGFβ1 at 96 h. (A) Traction maps of HCC1954 cells, (B) Cell area, (C) total force

exerted, (D) traction stress, and (E) net contractile moment of HCC1954 cell. .............. 36

vii

ACKNOWLEDGEMENTS

I owe my deepest gratitude to my family for giving me the opportunity to pursue higher

studies at The Pennsylvania State University. They have helped and supported me in every way

possible towards achieving my career goals.

I’m very grateful to my research advisor, Dr. Esther W Gomez, for her guidance and

support for pursuing my thesis work and following my career dreams. She has given me lots of

opportunities to develop my scientific knowledge and improve my technical communication

skills. I definitely improved myself a lot and became a better researcher because of her. I couldn’t

have imaged a better adviser and mentor.

I would like to thank Dr. Manish Kumar and Dr. Phillip E. Savage for agreeing to be in

my defense committee. They were supportive and encouraging which helped to the successful

completion of my thesis.

I would like to thank my lab members, Joseph W. O’Connor, Joelle Khouri, Natalie

Morrissey and Todd Thompson for their help and advice for performing my experiments. They

were very helpful in finishing my experiments on time.

I would like to express my sincere gratitude to Dr. Wayne Cutis and Dr. Thomas Wood

for letting me use their instruments for my research

I would also like to thank my other lab members Matt Bierowski, Akanksha Gupta, Dan Ye,

Joseph Wokpetah and Steve Pohler for making the lab environment more lively and joyful to

work. Thanks are also due to my friends who helped me at every time to realize my dream come

true.

1

Chapter 1

Background

Myofibroblasts are cells found in the body that help in wound healing but when they are

activated in pathological contexts they can contribute to the formation of fibrosis and lead to

cancer progression [1-9]. During wound healing they exert large contractile forces on the

extracellular matrix (ECM) and secrete ECM components that help in the closure of the wound

[1, 2]. Similarly, in pathological context these factors can lead to fibrosis and ultimately to the

failure of organs [3-6]. When present near the tumor front, myofibroblasts secrete certain

cytokines that promote the metastasis of tumor cells [7-9]. Myofibroblasts can also attack the

implanted biomaterials leading to their failure [10]. Myofibroblasts can be formed through

fibroblasts differentiation or they can also be formed through epithelial mesenchymal transition

(EMT) process [1, 11, 12]. Here, we study the mechanisms underlying myofibroblast formation

through EMT.

Epithelial-mesenchymal transition

Myofibroblasts can develop from lung [13-16], kidney [17-19], and breast [20-22]

epithelial cells through EMT. Transcriptional programs governing EMT are well documented;

there is down-regulation of epithelial markers such as E-cadherin (E-cadherin) and cytokeratins,

and up-regulation of various mesenchymal markers such as N-cadherin (N-cad) and vimentin [23,

24]. In EMT cells loose intercellular contacts, acquire an elongated morphology, and display

increased motility (Figure 1-1) [25-29]. During this process there is a dramatic remodeling of the

cytoskeleton and formation of stress fibers [23, 24, 30]. Cells can further progress through EMT

2

to express alpha-smooth muscle actin (α-SMA) protein, which is a hallmark of myofibroblast

formation [31].

Figure 1-1 Epithelial mesenchymal transition is characterized by loss of cell-cell junctions,

acquisition of an elongated morphology and formation of stress fibers. Mesenchymal cells further

progress through EMT to form myofibroblasts.

Transforming growth factor (TGF)-β1 is an important cytokine that is present near

wound, cancer and fibrosis sites, which is known to induced EMT in epithelial cells [32-34].

TGFβ1 activates various Smad and non-Smad pathways; Smad pathway induces transcription

factors that include, dEF1/ZEB1, SIP1/ZEB2, and Snail/SNAI1, and non-Smad pathways include

ERK, p38 MAPK, RhoA, and Rac1, regulate cell fate during EMT [35]. In this study we have

induced EMT in normal murine gland epithelial cells (NMuMG) and Madin-Darby canine kidney

(MDCK) epithelial cells using TGFβ1, and we examined the roles of ERK and p38 in the

regulation of EMT.

3

Evolution of cell generated forces and cytoskeletal signaling during EMT

Transcriptional programs governing during EMT have been studied extensively,

However, less is known about how actin cytoskeletal remodeling and cell generated forces evolve

during EMT progression [36-40] . Cytoskeletal remodeling during EMT involves loss of thin

cortical actin filaments and formation of thick parallel stress fibers made up of actin filaments

[41, 42]. Factors regulating, cytoskeletal rearrangements and the impact of cytoskeletal signaling

on EMT progression is poorly understood. Furthermore, the evolution of cell generated forces

during myofibroblast formation through EMT has not been studied. In this thesis, we examine the

evolution of cell generated forces during myofibroblast formation through EMT using the traction

force microscopy (TFM) technique. In addition, we examine the role of key signaling molecules

in the regulation of cytoskeletal remodeling and force generation.

EMT involves upregulation of a variety of cytoskeletal associated proteins such as α-

SMA [31], tropomyosin isoforms [43, 44] and caldesmon [44]. Incorporation of α-SMA into

stress fibers plays an important role in increased contractility of myofibroblasts formed through

fibroblasts[45]. Tropomyosin, upon binding to actin, activate actomyosin ATPase and promote

stress fibers formation [46]. Several studies have shown that an increase in caldesmon expression

stabilizes stress fibers [47]; however, others have shown that overexpression of caldesmon can

disrupt stress fibers [48]. It has also been shown that phosphorylation of caldesmon at ser527

releases the inhibitory effect of caldesmon on actomyosin ATPase activity leading to stabilization

of stress fiber formation [49-51] . Here, for the first time we correlate the expression of these

actin binding proteins (α-SMA, caldesmon, phosphorylated caldesmon at ser527 (p-caldesmon)

and tropomyosin) with cytoskeletal rearrangement and cell generated forces during myofibroblast

formation through EMT.

4

Role of ERK and p38 MAPK in development of myofibroblasts through EMT

Activation of various pathways such as MAPK pathways, RhoA, and Rac1 are needed for

stress fiber formation during EMT [36-40, 52] . The role of ERK1/2 MAPK in regulation of EMT

and associated stress fiber formation varies with cell types. In some cell types, blocking ERK1/2

activation using pharmacological inhibitors blocked EMT, α-SMA expression, and stress fibers

associated with EMT, whereas, in other cell types the pharmacological inhibitors did not block

EMT and associated stress fibers formation [40, 53-55]. Here, we examine the impact of the ERK

signaling pathway on cytoskeletal rearrangement and force generation in NMuMG and MDCK

cells. We find that inhibition of ERK increased the expression of α-SMA and cell-generated

forces in NMuMG cells during TGFβ1 induced EMT. However, in MDCK cells, inhibition of

ERK decreased the expression of α-SMA and associated cell generated forces.

The p38 MAPK plays an important role during induction of EMT and associated

stress fibers formation [26, 56-58]. The p38 MAPK is a part of MKK3/6-p38MAPK-ATF2

pathway which is activated during TGFβ1 induced EMT [56]. The p38 MAPK inhibitors such as

SB203580 and SB202190 (specific p38MAPK inhibitors) block the stress fibers formation and

change in cell morphology during EMT [56]. In NMuMG cells blocking p38 MAPK activation

has been shown to block the EMT characteristics such as loss of E-cadherin, acquisition of a

spindle-shaped fibroblast morphology, and formation of actin stress fibers [26]. In this

study we show the effect of p38 MAPK inhibitor, SB203580, on cell generated forces

and expression of actin binding proteins at transcript levels.

5

Chapter 2

Evolution of cell generated forces

Introduction

Evolution of cell generated forces during TGFβ1 induced EMT at 24, 48 and 72h were

studied on 1.8 KPa gels, which is comparable to the stiffness of an average breast tumor [59]. 1.8

KPa gels were also necessary because optimal beads movement was observed on this stiffness

during TFM technique which was used to measure the forces exerted by the cells. Hence, all the

experiments used in this study were conducted on 1.8 KPa gels. The total number of focal

adhesions and total focal adhesions area per cell was calculated at these time points. Phalloidin

staining of actin filaments was used to show stress fibers formation during myofibroblasts

formation through EMT. The expression of actin binding proteins caldesmon, p-caldesmon,

tropomyosin and α-SMA were also studied at these time points and correlated with cell generated

forces.

Materials and Methods

Cell culture

Normal murine mammary gland (NMuMG) epithelial cells were obtained from American Type

Culture Collection (ATCC CRL-1636) and were cultured in DMEM supplemented with 10% fetal

bovine serum (FBS; Atlanta Biologicals), 10 μg/ml insulin (Sigma), and 50 μg/ml gentamicin

(Life Technologies). Madin-Darby canine kidney (MDCK) epithelial cells were obtained from

Celeste Nelson (Princeton University) and were cultured in Eagle’s Minimum Essential Medium

6

supplemented with 10% FBS and 50 µg/ml gentamicin. Cells were grown in their culture media

in a 37°C humidified incubator with 5% CO2. For all experiments, cells were serum starved

overnight (2% serum in culture medium) and then treated with 10 ng/ml recombinant human

TGFβ1 (R&D Systems) or carrier vehicle (control) (1mg/ml BSA and 4mM HCl in H2O). For

inhibitor studies, cells were treated either with MEK1/2 inhibitor U0126 (10 μM; VWR) or with

p38 MAPK inhibitor SB203580 (10 μM; VWR) diluted in dimethyl sulfoxide (DMSO).

Preparation of substrates

Polyacrylamide (PA) gels with a modulus comparable to that of a mammary tumor (1.8

kPa) were fabricated following a modified protocol [60, 61]. Glass slides were incubated in 0.1N

sodium hydroxide (NaOH) for 15 minutes, thoroughly rinsed with water, and dried in air. The

slides were then incubated in 2% (v/v) aminopropyl trimethoxysilane (APTMS) diluted in

acetone for 30 minutes and were then rinsed with acetone and dried in a fume hood. Next, the

treated slides were incubated in 0.5% glutaraldehyde diluted in 1× phosphate buffered saline

(PBS) for 30 minutes followed by thorough washing with deionized (DI) water before drying in

air. A solution of 5% acrylamide, 0.06% bis-acrylamide, and DI water was degassed for 30

minutes before adding 0.5% of ammonium persulphate (10% w/v) and 0.05% of

tetramethylethylenediamine (TEMED) to initiate polymerization. This mixture was pipetted onto

the treated glass slide followed by placing a cover slip treated with Rain-X on the top of the

solution. Polymerization occurred over a 30 minute time frame.

Following the polymerization, PA gel surfaces were treated with 0.5 mM N-

Sulfosuccinimidyl-6-(4'-azido-2'nitrophenylamino) hexanoate (sulfo-SANPAH; Pierce) diluted in

50 mM HEPES buffer (pH 8.5) and the gel surfaces were exposed to ultraviolet light (CL-1000

Ultraviolet Crosslinker; UVP) for 10 minutes. The activation step was then repeated. After

7

activation, the PA gels were washed three times in 50 mM HEPES before incubation with10

μg/ml of human plasma fibronectin (BD Biosciences) for 2 hours at room temperature. The gels

were then washed three times with 50 mM HEPES buffer to remove excess fibronectin solution.

The PA gels were finally incubated with DMEM supplemented with 2% FBS for 1 hour in an

incubator prior to plating cells for experiments.

Western Blotting

Total protein was extracted from cells cultured on PA gels in the presence of TGFβ1 or

control vehicle for 24, 48, or 72 hours. Cells were washed twice with ice cold 1× PBS and then

lysed with ice cold RIPA buffer (Pierce) supplemented with protease and phosphatase inhibitors

(Pierce). The lysate was then centrifuged at 14000 rpm for 15 minutes at 4 ºC. The supernatant

was collected and the protein concentration was measured using a BCA Protein Assay Kit

(Pierce). Equal amounts of lysate were loaded and separated on a NuPAGE Novex 4-12% Bis-

Tris Protein Gels (Life Technologies) and transferred to a PVDF membrane. Membranes were

blocked with 5% non-fat dry milk and analysis was performed with primary antibodies against E-

cadherin (1:1000, Cell Signaling), αSMA (1:2500; Sigma), p-caldesmon (1:700; Abcam),

caldesmon (1:10000; Abcam), tropomyosin (1:700; Sigma), α-tubulin (1:1000, Sigma), ERK 1/2

(1:1000, Cell Signaling), and phosphorylated ERK 1/2 (1:1000, Cell Signaling) . Membranes

were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies

(1:2000; Cell Signaling) and SuperSignal West Pico Chemiluminescent Substrate (Pierce) for

detection. The blots were then imaged using FluorChem FC2 system (Cell Biosciences).

8

Traction force microscopy

Double-layer PA gels for traction force microscopy were fabricated as described

previously [62]. Briefly, a thin polyacrylamide gel (1800 Pa) without fluorescent beads was

attached to a gluteraldehyde treated coverslip as described above. Then, a second layer of PA gel

containing fluorescent beads (0.2 µm diameter; Life Technologies) was polymerized on top of the

first PA gel layer. Fibronectin was then cross-linked to the PA gel surface through the use of

sulfo-SANPAH chemistry.

NMuMG cells were seeded at 4000 cells/cm2 on PA gels embedded with fluorescent

beads. Experiments were conducted on an inverted epifluorescence microscope (Nikon Eclipse

TiE) equipped with a LiveCell environmental control chamber (Pathology Devices Inc.). Phase

contrast images of cells and fluorescent images of beads embedded in the gel before and after

removing the cells from the PA gel surface were collected with a 20× air objective. Bead

registration of the two fluorescent images was carried out by aligning the images with ImageJ

[63] and the TurboReg plug-in [64]. The displacements of the beads were determined and

traction stress maps were generated using LIBTRC 2.4 software (generously provided by

Professor Micah Dembo at Boston University) [65]. LIBTRC was also used to compute cell area,

total force |F|, average traction stress |T|, and net contractile moment.

Immunofluorescence staining

Samples were fixed at various time points with 4% paraformaldehyde in 1x PBS at room

temperature for 15 min, except for α-SMA which is fixed with 1:1 methanol/acetone at -20 °C for

10 min. After fixing, the cells were washed three times with 1x PBS, permeabilized with 0.1%

Triton X-100, blocked with 10% goat serum (Sigma) and were incubated with primary antibodies

9

for 1 h: α-SMA (1A4, Sigma), tropomyosin (TM311, Sigma), caldesmon (E89, Abcam) and E-

cadherin (Cell Signaling). Cells were then washed with 1x PBS 3 times before incubating them

with Alexa Fluor-conjugated secondary antibodies (Life Technologies). Following rinsing cells

were washed with 1x PBS 3 times and nuclei were counterstained with Hoechst 33342 (Life

Technologies).

For vinculin and actin staining, cells were fixed with 4% paraformaldehyde in 1x PBS,

cell were washed twice with wash buffer (0.05% Tween-20 in 1x PBS) and permeabilized with

0.1% Triton X-100 in 1x PBS for 5 min at room temperature. Cells were then washed twice with

wash buffer followed by blocking with 1% BSA solution in 1x PBS for 30 min at room

temperature. Anti-vinculin (V9131, Sigma) was diluted (1:100) to a working concentration in the

blocking solution and cells were incubated with antibody for 1 h at room temperature followed by

washing with wash buffer three times. Cells were then incubated with Alexa Fluor-conjugated

secondary antibodies (1:100, Life Technologies) in 1x PBS for 60 min at room temperature. To

stain for actin, cells were incubated with Alexa Fluor 594 phalloidin (1:250, Life Technologies)

simultaneously with the secondary antibody for 60 min at room temperature. Samples were then

washed three times with wash buffer. Following washing, nuclei were counterstained with

Hoechst 33342 (Life Technologies) at room temperature for 2 min, followed by washing three

times with wash buffer. All samples were then mounted on cover glass slide with Fluormount-G

(Electron Microscopy Sciences).

Microscopy and Focal Adhesion Analysis

Samples were imaged using a 20x or 40 x air objectives on a Nikon Eclipse Ti-E inverted

fluorescence microscope equipped with a Photometrics CoolSNAP HQ2 CCD camera. To

determine focal adhesion properties, stack containing vinculin images of single cells were

10

uploaded to ‘The Focal Adhesion Analysis Server’ (FAAS) [66]. Briefly, free hand tool in

ImageJ software is used to draw a boundary along a single cell per image to isolate it from group

of cells present in the vinculin immunofluorescence images taken at 40x by using clear outside

command in the edit menu. After all the single cells isolated from images were saved, they were

stacked using ImageJ and saved in ‘Tiff’ format which is uploaded to the FAAS. For the analysis

in the FAAS, detection threshold is set as 2 and minimum focal adhesions size is set as 10 pixels2,

which is equivalent to 0.256 µm2. Also, ‘Only Calculate Static Properties’ option is selected since

these are the static images. The FAAS gives the number of focal adhesions per cell and area of

each focal adhesion. This data was then used to plot histograms of the number of focal adhesions

per cell and the total area of all focal adhesions per cell using different treatment conditions.

Minimum of 50 cells were analyzed per sample condition over three different experiments.

Statistical Analysis

A minimum of three independent trials were performed for all experiments. A student’s t-test or

analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test for multiple comparisons

were used for sample comparisons using Kaleidagraph 4.1 software. Differences were considered

significant for p < 0.05.

Results

To examine how TGFβ1 regulates EMT and actin binding proteins expression,

immunofluorescence staining and western blot were performed

Immunofluorescence staining at 24, 48 and 72 h on NMuMG cells treated with control

and TGFβ1 was performed for E-cadherin, α-SMA, caldesmon, and tropomyosin. Staining for

11

the epithelial marker E-cadherin showed that there is reduced expression of E-cadherin at cell-cell

junctions in TGFβ1 treated samples compared to controls at 24 , 48 and 72 h. α-SMA was found

to be expressed at 72 h post the treatment of cells with TGFβ1 but not in control treated cells,

which suggests that myofibroblasts form at 72 h. Caldesmon and tropomyosin were upregulated

in TGFβ1 treated cells at 24 , 48 and 72 h compared to control cells, also, these proteins seemed

to localize to stress fibers as seen in the figure Figure 2-1.

Figure 2-1 TGFβ1induces changes in epithelial and mesenchymal proteins. NMuMG cells treated

with control and TGFβ1 are stained for E-cadherin, α-SMA, tropomyosin, and caldesmon at 24,

48 and 72 h. Scale bar: 25 μm.

Western blot showed that the expression levels of caldesmon, p-caldesmon, and

topomyosin are increased in NMuMG cells at 24 h after the treatment with TGFβ1 compared to

control vehicle treated cells. The expression levels of these actin binding proteins remained

12

elevated at 48 h and 72 h in TGFβ1 treated cells. The expression of α-SMA was observed at 72 h

post treatment with TGFβ1, which suggests the formation of myofibroblasts at 72 h.

Figure 2-2 Western blot of control and TGFβ1 treated cells at 24, 48 and 72 h for E-cadherin, α-

SMA, tropomyosin, p-caldesmon, caldesmon, and α-tubulin, the loading control. The expression

of actin binding proteins caldesmon, p-caldesmon and tropomyosin is upregulated during TGFβ1

induced EMT and α-SMA is expressed at 72 h following the treatment with TGFβ1.

Immunofluorescence staining for vinculin and phalloidin staining for filamentous actin

was performed on cells treated with control vehicle or TGFβ1 at 24, 48 and 72 h. Thick, parallel

actin filaments formed in TGFβ1 treated cells at all the time points, whereas control treated cells

showed cortical actin around the cell peripheries (Figure 2-3). Focal adhesion analysis on cells

stained with vinculin showed that there is an increase in the total number of focal adhesions and

in the total focal adhesion area per cell from 24 h to 72 h after the treatment of NMuMG cells

with TGFβ1 (Figure 2-4). Also, TGFβ1 treated samples showed higher total focal adhesion area

13

and more focal adhesions per cell when compared to corresponding control treated cells at all the

time points. As seen from the immunofluorescence images there is an increase in the size of focal

adhesions at 72 h time point in TGFβ1 treated samples, which correlates with increased α-SMA

expression.

Figure 2-3 During the progression of EMT the actin cytoskeleton rearranges from a cortical

architecture to prominent stress fibers. Focal adhesion number and area increase during the

progression of EMT. Scale bar: 25 μm.

Traction maps generated from TFM analysis on NMuMG cells show that there is increase

in traction stresses across the cells as they undergo TGFβ1 induced EMT (Figure 2-3. A). TFM

analysis also showed that there is increase in area, total forces, and net-contractile moment

(measure of over-all contractility of the cell) in the cells from 24h to 72 h in TGFβ1 treated

samples (Figure 2-3 B, C & D). The increased expression of forces in TGFβ1 treated cells at 72 h

correlates compared to 24 h and 48 h correlate with expression of α-SMA and increase in number

and total area of focal adhesions, which was observed in immunofluorescence staining and

western blots. Also at each time point control treated cells have shown less area, total forces, and

net-contractile moment compared to TGFβ1 treated cells (Figure 2-3 B, C & D).

14

Figure 2-4 Cells exhibit increased focal adhesions following induction of EMT. (A)

Immunofluorescence staining for vinculin. Scale bar: 25 μm. Quantification of (B) focal adhesion

number and (C) focal adhesion area.

15

Discussion

NMuMG cells cultured on 1.8 kPa gels show increased expression of caldesmon, p-

caldesmon, and tropomyosin upon treatment with TGFβ1 as cells undergo EMT. After initial

upregulation of these actin binding proteins, expression of α-SMA is observed at 72 h. Previous

studies showed that an increase in cellular tension is necessary for the induction of αSMA

expression during formation of myofibroblast through fibroblast differentiation [67]; however,

this has not yet been demonstrated during myofibroblast formation through EMT. Here, using

TFM, we show that the tension in cells is increased before expression of α-SMA and there is

increase in focal adhesions size in TGFβ1 induced EMT. Also, it is known that, during

myofibroblast formation through fibroblasts the expression of α-SMA actin increases the size of

focal adhesions and cell generated forces [45, 68]. Even in our experiments we show that the

size of focal adhesions and traction forces increase at 72 h time point correlating with α-SMA

expression.

Traction force microscopy was also performed on MDCK cells at 72 h post the treatment

with control and TGFβ1 treatment with 1.8 kPa gels. Traction maps show traction stresses

increase along the cell during TGFβ1 induced EMT compared to control treated cells (Figure 2-6

A). Average area, total force exerted, net contractile moment and traction stress, per cell,

increased with TGFβ1 treatment compared to control treated cells (Figure 2-6 B, C, D, & E).

16

Figure 2-5 TFM analysis of NMuMG cells (A) Maps of traction stresses along the cells. Average

(B) area, ##p<0.001 and #p<0.0001, *p<0.001 in comparison to 24 h TGFβ1 (C) force, # p <

0.0001; *** p < 0.0001 in comparison to 24 h and 48 h TGFβ1 (D) net contractile moment # p <

17

0.000001; *** p < 0.01 in comparison to 24 h and 48 h TGFβ1 and (E) traction stress #p<0.5,

##p<0.0001 and *p<0.01 in comparison to all other samples.

Figure 2-6 TFM analysis of MDCK cells treated with control or TGFβ1 at 72 h. (A) Maps of

traction stresses along the cells. Average (B) area (C) force (D) net contractile moment and (E)

traction stress. *p<0.0001 in comparison to respective control samples.

18

Chapter 3

Role of ERK ½ and p38 MAPK in acquisition of myofibroblast characteristics

Introduction

TGFβ1 activates various MAPK pathways that include ERK1/2 and p38 MAPK

pathways [26, 40, 53-58]. ERK1/2 MAPK is a part of Ras/Raf/MEK/ERK pathway, whereas p38

MAPK is a part of MKK3/6-p38MAPK-ATF2 pathway [56, 69]. Studies show P38 MAPK is

necessary for TGFβ1 induced EMT, whereas role of ERK in EMT varies with cell types [26, 56,

70]. How these MAPK pathways regulate myofibroblast characteristics during EMT is not

studied. We used U0126, a specific pharmacological inhibitor for MEK1/2 which blocks the

activation of MEK1/2 which in turn abolishes the phosphorylation of ERK1/2, to study the effect

of the ERK1/2 pathway in acquisition of myofibroblast characteristics. Also for blocking the

activation of p38 MAPK we have used SB203580 inhibitor to study traction forces and transcript

levels of actin binding proteins during TGFβ1 induced EMT.

Results

Phosphorylation of ERK1/2 was studied at 24 , 48 and 72 h following the treatment of

cells with TGF𝛽1 or control vehicle using western blot, which showed that there’s no notable

upregulation of ERK1/2 phosphorylation during myofibroblasts formation through EMT in

NMuMG cells at 48 and 72 h (Figure 3-1. A). At 24 h the ERK1/2 phosphorylation seems

upregulated in TGFβ1 treated samples compared to control treated samples.

19

Figure 3-1 Western blot showing (A) ERK1/2 phosphorylation at 24, 48 and 72 h in TGFβ1 and

control treated NMuMG cells (B) ERK phosphorylation upon treatment with TGFβ1 + U0126

and TGFβ1+dmso at 72 h (C) ERK phosphorylation, expression of α-SMA, tropomyosin,

caldesmon and p-caldesmon at 72 h in NMuMG cells treated with control/TGFβ1 plus

DMSO/U0126, also shown are α-SMA, tropomyosin, caldesmon and p-caldesmon.

Cells were treated with MEK1/2 specific inhibitor, U0126, which blocked

phosphorylation of ERK1/2 (Figure 3-2. B&C). Treatment of U0126 increased the expression

of α-SMA in TGFβ1 treated samples and there is no expression of α-SMA in control plus

DMSO or U0126 treated samples (Figure 3-1 B & C). Western blot was performed in

samples treated with TGFβ1 plus DMSO or U0126 treated samples at 24, 48 and 72 h on

NMuMG cells. ERK1/2 phosphorylation was blocked at all the time points in TGFβ1

plus U0126 treated samples. In TGFβ1 plus U0126 treated samples, initially, there is

reduced expression of caldesmon, p-caldesmon, and tropomyosin at 24 h time point

20

compared to TGFβ1 plus DMSO treated samples, but at 48 h and 72 h, the expression

levels of these proteins was similar in both TGFβ1 plus DMSO and U0126 treated

samples (Figure 3 1. B).

Figure 3-2 Real time - PCR at 72 h in NMuMG cells treated with control or TGFβ1 plus DMSO

or U0126. Transcript levels for (A) E-cadherin, **p<0.001, #p<0.05, (B) α-SMA, *p<0.01,

#p<0.5 compared to TGFβ1 plus DMSO, (C) caldesmon, #p<0.5, *p<0.01 compared to control

plus DMSO, and (D) tropomyosin, *p<0.01, #p<0.5 compared to control plus DMSO.

The expression levels of caldesmon, p-caldesmon, and tropomyosin remained

upregulated in TGFβ1 plus DMSO or U0126 treated samples compared to control plus DMSO

21

treated sample. Interestingly, control samples treated with U0126 also showed expression up-

regulation of caldesmon, p-caldesmon, and tropomyosin compared to control plus DMSO treated

samples at 72 h (Figure 3 1. C).

Real time- PCR shows that transcript levels of EMT marker E-cadherin is downregulated

in both TGFβ1 plus DMSO or U0126 samples compared to control plus DMSO or U0126 (Figure

3-2 A). α-SMA, caldesmon, and tropomyosin transcript levels are upregulated with U0126

treatment in TGFβ1 treated samples at 72 h in NMuMG cells (Figure 3-2 B, C & D). The

transcript levels of α-SMA are more in TGFβ1 plus U0126 treated samples compared to TGFβ1

plus DMSO treated samples (Figure 3-2 B). The transcript levels of caldesmon and tropomyosin

are more in TGFβ1 plus DMSO or U0126 samples compared to control plus DMSO or U0126

treated samples (Figure 3-2 C &D).

Figure 3-3 Staining for actin at 72 h in NMuMG cells treated with control or TGFβ1 plus DMSO

or U0126. U0126 did not block TGFβ1 induced stress fibers formation at 72 h. Scale bar ~ 25μm.

F-actin and vinculin staining was performed on NMuMG cells treated with control

vehicle or TGFβ1 plus DMSO or U0126 at 72 h. TGFβ1 plus DMSO or U0126 treated samples

showed actin stress fibers indicating that U0126 did not block TGFβ1 induced stress fibers

formation at 72 h (Figure 3-2). The total focal adhesion area and total focal adhesion number per

22

cell increased in TGFβ1 plus U0126 treated cells when compared to TGFβ1 plus DMSO treated

cells and control plus DMSO or U0126 treated cells (Figure 3 3).

Figure 3-4 U0126 did not block TGFβ1 induced focal adhesions formation at 72 h. (A)

Immunofluorescence staining for vinculin at 72 h in NMuMG cells treated with control or TGFβ1

plus DMSO or U0126, and quantification of (B) focal adhesion area and (C) focal adhesion

number.

Traction force microscopy revealed a significant increase in cell area, forces exerted, and

net contractile moment by the TGFβ1 plus U0126 treated cells compared with TGFβ1 plus

23

Figure 3-5 Traction force microscopy at 72 h post the treatment of NMuMG cells treated with

control/TGFβ1 plus DMSO/U0126 was used to generate (A) traction stress maps, and to calculate

24

(B) cell area, #p<0.0001, *p<0.0001 comparison to TGFβ1 + DMSO, (C) force exerted by the

cells, ##p<0.001, #p<0.0001, *p<0.0001 comparison to TGFβ1 + DMSO, (D) net contractile

moment of the cells, #p<0.0001, *p<0.0001 comparison to TGFβ1 + DMSO, and (E) average

traction stress exerted by the cells, p<0.05 in comparison to control plus DMSO. N=50 cells per

condition and ##p<0.001, #p<0.0001, *p<0.0001 comparison to TGFβ1 + DMSO.

DMSO treated cells (Figure 3-4 A, B, C, & D). Also, TGFβ1 plus DMSO or U0126 treated cells

have a larger area, exert more forces and have more net contractile moment compared to control

plus DMSO or U0126 treated cells, respectively. This increased in forces exerted by TGFβ1 plus

U0126 treated cells correlates with increased expression of α-SMA, and an increase in total focal

adhesion area and total focal adhesion number per cell compared to TGFβ1 plus DMSO treated

cells (Figure 3-1 and Figure 3-3).

For NMuMG cells, treatment with U0126 enhances the acquisition of myofibroblast

characteristics during EMT, including increased levels of α-SMA and increased cell generated

traction forces. Next, we used MDCK cells to examine the role of ERK1/2 signaling in increasing

myofibroblast characteristics is cell type specific. Western blot analysis showed that ERK1/2

phosphorylation was blocked in all TGFβ1 and control plus DMSO and U0126 treated samples at

the 72 h time point (Figure 3-5). Interestingly, ERK 1/2 phosphorylation was increased in TGFβ1

plus DMSO treated cells compared to control plus DMSO treated cells. TGFβ1 treated cells

showed reduced expression of α-SMA when treated with U0126 compared to DMSO treated cells

(Figure 3-5). Furthermore, TGFβ1 induced high contractile forces at 72 h in MDCK cells

compared to control treated cells (Figure 2-6). TFM analysis was also performed at 72 h with

control or TGFβ1 plus DMSO or U0126 to study the effect of ERK1/2 signaling on cells

generated forces. Inhibition of EKR1/2 phosphorylation reduced cell generated traction forces

(Figure 3-6). These results show that U0126 inhibition of ERK1/2 activation results in reduced

myofibroblast characteristics in MDCK cells.

25

Figure 3-6 Western blot showing expression of p-ERK1/2, ERK1/2, α-SMA, E-cadherin, and α-

tubulin, the loading control, in MDCK cells at 72 h treated with control or TGFβ1 plus DMSO or

U0126.

Traction force microscopy was performed with NMuMG cells using p38 MAPK inhibitor

SB203580 during TGFβ1 induced EMT (Figure 3-7). The area of cells treated with TGF β1 plus

SB203580 increased compared to that of control plus SB203580, but when compared with TGF

β1 plus DMSO treated cells, the TGF β1 plus SB203580 cells showed less cell area (Figure 3-7

A). Total forces in cells treated with TGF β1 plus SB203580 appeared to be same when compared

with TGF β1 plus DMSO (Figure 3-7 B). Net contractile moment of TGF β1 plus SB203580

decreased when compared to TGF β1 plus DMSO treated cells (Figure 3-7 C). There were no

significant differences in average traction stresses exerted by the cells when treated with TGF β1

plus SB203580 or DMSO (Figure 3-7 D).

26

Figure 3-7 Traction force microscopy at 72 h post the treatment of NMuMG cells treated with

control or TGFβ1 plus DMSO or SB203580. (A) cell area, (B) force exerted by the cells, (C)

net-contractile moment of the cells, and (D) average traction stress exerted by the cells; #p<0.05

and *p<0.0001.

Real time-PCR was also performed with inhibitor SB203580 on NMuMG cells during

TGFβ1 induced EMT (Figure 3-8). E-cadherin transcript levels in TGF β1 plus SB203580

decreased compared to control plus DMSO treated samples (Figure 3-8 A). Transcript levels of α-

27

SMA, caldesmon and tropomyosin appeared same in TGF β1 plus SB203580 treated samples

when compared with TGF β1 plus DMSO treated samples (Figure 3-8 B, C, and D).

Figure 3-8 Real time - PCR at 72 h in NMuMG cells treated with control or TGFβ1 plus DMSO

or SB203580. Transcript levels for (A) E-cadherin, *p<0.001, (B) α-SMA, (C) caldesmon, and

(D) tropomyosin.

28

Discussion

Blocking of ERK phosphorylation has been shown to block EMT in some cell types and

does not have any effect in other cell types [26, 56]. In renal tubular cells it was shown that

treatment with a MEK inhibitor blocks actin fiber formation and α-SMA synthesis [54].

Here, we show that blocking ERK phosphorylation increases the number and total area of

focal adhesions, the α-SMA expression, and cell generated forces in NMuMG cells.

However blocking ERK activation in MDCK cells led to downregulation of α-SMA and

cell generated forces. Hence, the results here show that ERK might play different role in

acquisition of myofibroblast characteristics in different cell types. Inhibition of ERK1/2

signaling could be a useful strategy in abrogating myofibroblast activation in pathological

contexts or for activating them in wound healing contexts, but how the ERK pathway

behaves in different epithelial cells types in guiding them towards myofibroblast should

be further studied.

29

Figure 3-9 Traction force microscopy at 72 h on MDCK cells treated with control or TGFβ1 plus

DMSO or U0126. (A) traction maps showing traction stress across the cells, (B) cell Area,

30

#p<0.001 and *p<0.001 compared to TGFβ1 plus DMSO, (C) forces exerted by the cells,

#p<0.001 and *p<0.001 compared to TGFβ1 plus DMSO, (D) net contractile moment of the cells,

#p<0.001 and ##p<0.05, and (E) average traction stress exerted by the cells, #p<0.001 and

##p<0.05. Minimum of 50 cells were used per condition over three different experiments.

31

Chapter 4

Regulation of focal adhesion proteins expression during epithelial

mesenchymal transition of HCC1954 cells with matrix stiffness

Introduction

Lipoma-preferred partner (LPP) and vinculin are proteins that are recruited to focal

adhesions during TGFβ1 induced EMT [71, 72]. This chapter discusses the focal adhesion

properties and forces exerted by the cells during TGFβ1 induced EMT in Human mammary gland

epithelial cells (HCC1954). The expression levels of focal adhesion proteins including vinculin

and LPP are studied on gels of varying stiffness during TGFβ1 induced EMT in HCC1954 cells.

LPP is known to localize to focal adhesions during TGFβ1 induced EMT and help in the

promotion of carcinoma invasion [73]. LPP is shown to regulate migration and invasion of breast

cancer cells during TGFβ1 induced EMT; TGFβ1 re-localizes LPP to focal adhesion complexes,

which is critical for TGFβ1 mediated focal adhesion turn over [71]. During EMT LPP also acts

as a sensor for extracellular signals promoting invasiveness of tumor cells [74]. Vinculin is a

protein that is recruited from cytoplasm to focal adhesion during TGFβ1 induced EMT [72].

Actin filaments converge into the focal adhesion complexes where they associate to FN receptors,

α5β1 integrin and bind to vinculin [75]. Here we showed that cancer cells acquired matured focal

adhesions on treatment with TGFβ1, and since matured focal adhesions are known to increase

cell generated forces, traction force microscopy analysis was performed on HCC1954 cells during

TGFβ1 induced EMT to check for modulation of cell generated forces.

32

Materials and Methods

Human mammary gland epithelial cells (HCC1954) were purchased from American Type

Culture Collection (ATCC) and maintained in RPMI 1640 media supplemented with 10% fetal

bovine serum (FBS; Atlanta Biologicals) and 50 µg/ml gentamicin (Life Technologies). Cells

were grown in the culture media in a 37°C humidified incubator with 5% CO2. For all

experiments, cells were serum starved overnight (2% serum in culture medium), and then treated

with 2.5 ng/ml recombinant human TGFβ1 (R&D Systems) or carrier vehicle (control) (1mg/ml

BSA and 4mM HCl in H2O) for 96 h.

For the following methods refer to chapter 2, materials and methods section for detailed

protocol. Cells were cultured on polyacrylamide gels of various stiffness’s including 130 Pa,

1800Pa, 5000 Pa and 6000 Pa. Gels were activated with sulfo-SANPAH, and for western blot

experiments 300,000 cells were added to the gels, for immunofluorescence experiments 150,000

cells and for TFM experiments 50,000 cells were added per sample. For performing TFM

analysis 5 kPa polyacrylamide gels were used. For western blot experiments and

immunofluorescence experiments cells were cultured on 130 Pa, 1800Pa, 5000 Pa and 6000 Pa

polyacrylamide gels.

Immunofluorescence staining for vinculin staining was performed using the same

protocol that was followed in chapter 2. For LPP staining cell signaling staining protocol is

followed, briefly, 4% paraformaldehyde fixed samples were washed with 1x PBS followed by

blocking in blocking buffer (1x PBS / 5% normal serum / 0.3% Triton™ X-100) for 60 min. LPP

(Cell Signaling) primary antibody was diluted (1:100) in antibody dilution buffer (1x PBS / 1%

BSA / 0.3% Triton™ X-100) and added to the samples after aspirating the blocking solution.

Samples were incubated overnight at 4°C and rinsed three times in 1x PBS for 5 min each.

Specimen were incubated in fluorochrome-conjugated secondary antibody (goat anti mouse)

33

diluted in antibody dilution buffer for 1 h at room temperature in the dark. Following washing,

nuclei were counterstained with Hoechst 33342 (Life Technologies) at room temperature for 2

min, followed by washing three times with wash buffer. All samples were then mounted on cover

glass slide with fluormount-G (Electron Microscopy Sciences).

Method for performing western blot used is same as that mentioned in chapter 2. For

western blot vinculin is used at dilution of 1:500 and LPP is used at dilution of 1:1000. GAPDH

is used as loading control at dilution of 1:1000.

Results

Western blot analysis shows that stiffness of extracellular matrix regulates the expression

of vinculin and LPP in low serum medium (2% FBS). Both vinculin and LPP are upregulated at

higher stiffness of 6000 Pa compared to 130 Pa or 1800 Pa gels in both control and TGFβ1

treated samples at 96 h (Figure 4-1 A). But, in high serum medium (10% FBS) there was no

difference of levels of vinculin and LPP expression on 130 Pa and 6000 Pa gels (Figure 4-1 B).

34

Figure 4-1 Western blot showing expression of vinculin, LPP, and GAPDH, the loading control,

in HCC1954 cells at 96 h treated with control or TGFβ1 in (A) low serum medium and (B) high

serum medium.

Figure 4-2 Immunofluorescence staining for vinculin in HCC1954 cells at 96 h post the treatment

of cells with control or TGFβ1 on 130 Pa, 1800 Pa and 6000 Pa gels.

35

Immunofluorescence staining for vinculin protein also shows that TGFβ1 increases the

induction of vinculin protein to focal adhesions on all the gels of 130 Pa, 1800 Pa and 6000 Pa

stiffness (Figure 4-2). Also, the cells grown on higher stiffness of 6000 Pa appeared to have the

higher number of focal adhesions compared to cells grown on lower stiffness of 130 Pa and 1800

Pa in TGFβ1 Treated samples.

Traction force microscopy analysis at 96 h on control and TGFβ1 treated samples show

that the traction stress in the cells increase with TGF β1 treatment compared with control treated

samples (Figure 4-2 A). Cell area of HCC1954 cells, and total force exerted, traction stress and

net contractile moment of HCC1954 cells increased with TGF β1 treatment compared to control

treated cells on 5000 Pa gels (Figure 4-2 B, C, D & E).

Conclusion

During TGFβ1 induced EMT the upregulation of vinculin and LPP were found to be

related to the stiffness of extracellular matrix on which the cells were growing in low serum

medium but not in high serum medium. The expression of these focal adhesion proteins increased

in 6000 Pa gels compared to 1800 Pa and 130 Pa gels. Traction force microscopy was performed

with HCC1954 showed that cell area, total forces exerted by the cells, net contractile moment and

traction stress exerted by the cells increased during TGF β1 induced EMT at 96 h correlating with

increased expression of focal adhesion proteins.

36

Figure 4-3 Traction force microscopy analysis of HCC1954 cells treated with control or TGFβ1

at 96 h. (A) Traction maps of HCC1954 cells, (B) Cell area, (C) total force exerted, (D) traction

stress, and (E) net contractile moment of HCC1954 cells. Minimum of 50 cells over 3 different

experiments were used per condition, #p < 0.01, *p < 0.0001 when compared to control treated

samples.

37

Chapter 5

Conclusion and Future

TFM was used to show the evolution of cell generated forces during EMT for the first

time. In this study we have shown that cell generated forces increase before expression of α-SMA

by NMuMG cells. The expression of α-SMA further correlates with increased contractile activity

of the cells, and total number and area of focal adhesion per cell. Previous it was known that

expression of α-SMA in myofibroblasts formation through α-SMA negative fibroblasts cells

required certain amount of cellular tension, and expression of α-SMA further increases the size of

focal adhesions, but this was not show in myofibroblasts formation through EMT [45, 55]. The

results obtained in this study show that this similar pattern is observed in myofibroblast formation

through EMT as seen in myofibroblast formation through fibroblast. However, blocking α-SMA

protein expression and seeing how it affects the cell generated forces, and focal adhesions size

and number, will further strengthen this theory.

ERK is known to have no effect on TGFβ1 induced EMT and stress fibers formation in

some cell types and blocks EMT in other cell types [26, 56]. In renal tubular cells it is shown that

TGFβ1 induced α-SMA and EMT is blocked during EMT [54]. However, how inhibiting the

ERK phosphorylation controls the expression of α-SMA and corresponding cell generated forces

have not been studied in detail. Here we have shown that inhibiting ERK in NMuMG cell

increases the myofibroblast characteristics, which are increased expression of α-SMA and cell

generated forces. We have also show that this increase in α-SMA is correlated with increase in

total focal adhesion area and number per cell. In contract, inhibiting ERK phosphorylation in

MDCK cell has downregulated the expression of α-SMA and cell generated forces. These results

38

show that ERK regulation of myofibroblasts development through EMT varies with different cell

types. This ERK regulation can be helpful in abrogating the myofibroblast in pathological

contexts or to activate the myofibroblast formation in reparative conditions.

Experiments with HCC1954 cells showed that cells upregulated vinculin and LPP with

stiffness in low serum medium but not in high serum medium. Traction force microscopy analysis

showed that cell area, total forces exerted by the cells, net contractile moment and traction stress

exerted by the cells increased during TGF β1 induced EMT. Further studies needed to be done to

know the area and number of focal adhesions per cell during TGFβ1 induced EMT to correlate

with cell generated forces.

39

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