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Effect of KRAS and mTOR inhibitors on tissue factor gene expression in two

human pancreatic cancer cell lines


Grace Lian

Senior Honors Thesis

Department of Biology

Mackman Lab—Department of Medicine (Division of Hematology/Oncology)

University of North Carolina at Chapel Hill

March 31, 2020



Dr. Nigel Mackman, Thesis Advisor

Dr. Wolfgang Bergmeier, Reader


Effect of KRAS and mTOR inhibitors on tissue factor gene expression in two

human pancreatic cancer cell lines

Grace Lian

Yohei Hisada and Nigel Mackman


Tissue factor (TF) is a transmembrane receptor and cofactor of factor (F) VII/VIIa. The TF/FVIIa complex activates the coagulation cascade. It is highly expressed in pancreatic ductal adenocarcinoma (PDAC). Elevated levels of circulating, tumor-derived TF+ extracellular vesicles are associated with venous thromboembolism in pancreatic cancer. Since hemostasis requires TF, systemic inhibition of TF to reduce VTE risk would lead to severe bleeding. Previous studies reported that the Kirsten rat sarcoma viral oncogene homolog (KRAS) and mammalian target of rapamycin (mTOR) pathways regulate TF gene expression in human cancer cells. Therefore, we hypothesized that blocking KRAS and mTOR would reduce TF gene expression in PDAC. We examined the effect of the KRAS inhibitor stabilized alpha helices of son of sevenless 1 (SAH-SOS1A) and the mTOR inhibitor sapanisertib on TF gene expression in two high-TF expressing PDAC cell lines HPAF-II and BxPC-3. We hypothesized that 1) inhibition of KRAS with SAH-SOS1A would reduce TF expression in HPAF-II cells, which contain a constitutively active mutant KRAS, to a greater extent than in BxPC-3, which contains wild-type KRAS, and that 2) mTOR inhibition with sapanisertib would reduce TF expression in both cell lines. HPAF-II and BxPC-3 were treated for 48 hours with either SAH-SOS1A or sapanisertib. Cell viability and growth was measured with a resazurin uptake assay, and total protein concentration was determined using Pierce BCA and DC assays to examine if the inhibitors were toxic. Levels of cellular TF activity were determined using a one-stage coagulation assay. Our study yielded an unexpected result: both inhibitors were toxic to the cells and reduced cell viability, total protein, and TF activity. Taken together, these preliminary studies suggest that the decrease in TF activity in cells treated with either inhibitor is due to cell death rather than a selective inhibition of TF gene expression.


Tissue factor (TF) is a transmembrane glycoprotein receptor that binds the serine

protease factor VIIa (FVIIa) to initiate the extrinsic blood coagulation cascade.1 The

TF:FVIIa complex also activates a variety of different cell-signaling pathways that


(PARs).2–4 Aberrant expression of TF has been detected in a range of malignancies5 and

is associated with cancer stage and poor prognosis.6,7

Pancreatic ductal adenocarcinomas (PDAC) express very high levels of TF.4,7–9

PDAC accounts for approximately 85-90% of pancreatic cancers8,10 and has a 5-year

survival rate of ~8%.4,8 Pancreatic cancer also has one of the highest rates of venous

thromboembolism (VTE) (5-26%),11–15 a leading cause of mortality in cancer patients.16– 22 Furthermore, elevated VTE rates are observed in pancreatic cancer patients with high

TF expression compared to those with low TF expression.4,23

Given these observations, targeting TF may reduce VTE and improve the survival

of patients with pancreatic cancer. However, because TF is crucial for hemostasis,

systemic inhibition of TF would be accompanied by an increased bleeding risk. Instead,

we hypothesize that reducing TF expression by targeting intracellular signaling pathways

that regulate TF gene expression in pancreatic tumors would reduce VTE without

bleeding in PDAC patients. This project sought to examine the effect of inhibiting two

major signaling pathways involved in cancer on TF gene expression in two human

pancreatic cancer cell lines. These pathways were 1) the Kirsten rat sarcoma viral

oncogene homolog (KRAS) pathway, and 2) the mammalian target of rapamycin (mTOR)

pathway. We used HPAF-II and BxPC-3 cell lines, both of which express high levels of


The first target is KRAS, which activates the rapidly accelerated fibrosarcoma

(RAF) kinase – mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) – extracellular

signal-regulated kinase (ERK) pathway through sequential phosphorylation.25 The


proliferation, and therefore when mutated is often implicated as a driver of cancer


KRAS activating mutations occur in ~94% of pancreatic cancers.25,28 KRAS

activation in the human colorectal carcinoma cell lines HCT116 and DLD-1 is associated

with increased TF expression.17 A retrospective study also revealed an association

between KRAS mutation and VTE risk in patients with metastatic colon cancer, and suggested that the increased VTE risk may be related to elevated TF expression.29

We determined the effect of stabilized alpha helices of son of sevenless 1

(SAH-SOS1A), a peptide inhibitor of both wild-type (WT) and mutant KRAS,26 on TF expression

in HPAF-II and BxPC-3 cells. SAH-SOS1A binds to the KRAS binding pocket with high

affinity and inhibits KRAS association with GTP while also preventing KRAS from

interacting with wild-type SOS1,26 a guanine nucleotide exchange factor that serves as

the catalyst for GTP activation of KRAS.26,27,30 We hypothesized that inhibition of KRAS

would lead to a greater reduction in TF in HPAF-II, which contains a mutant KRAS with

increased activity (KRAS G12D mutant24), than in BxPC-3 (KRAS WT9). This project also

investigated the effect of SAH-SOS1A on cell viability and total protein concentration, and

examined the phosphorylation of ERK, a downstream effector of KRAS, to confirm KRAS


The second part of this project focused on mTOR, a downstream serine/threonine

kinase essential to the PI3K/AKT/mTOR signaling cascade.31 mTOR is a key regulator of

cell growth and survival, metabolism, and migration,33–36 but its overexpression has been

implicated in the progression of several cancers.36 Studies have observed mTOR


We examined the effect of the mTOR inhibitor sapanisertib (also known as

MLN0128, TAK-228, and INK 128) on TF gene expression in HPAF-II and BxPC-3.

Sapanisertib is a drug that is currently being evaluated in clinical trials to treat patients

with various cancers,34 including pancreatic neuroendocrine tumors (PNET).40 It inhibits

the kinase activity of mTOR by competitively binding to the ATP site in the mTOR kinase

domain.34,41–44 It has been shown that inhibition of mTOR with sapanisertib reduces

mRNA levels and activity of TF in BON, a human PNET cell line, both in vitro and in vivo.41

This suggests that mTOR is an upstream regulator of TF in PNET.41 Therefore, we

expected that sapanisertib would yield similar regulatory effects on TF expression in our

PDAC cell lines. We also investigated its effect on cell viability and total protein


Overall, our goal was to determine if inhibition of the 1) KRAS and 2) mTOR

signaling pathways reduces TF gene expression in the PDAC cell lines HPAF-II and



Cell Culture

HPAF-II and BxPC-3 cells were cultured in T150 flasks with MEM (Thermo Fisher

Scientific, Waltham, MA, Cat#11095-080) and RPMI 1640 (Thermo Fisher Scientific,

Cat#11875-093), respectively, and were maintained at standard conditions (5% CO2,

37ºC). Both media contained 10% fetal bovine serum (FBS) (Omega Scientific, Tarzana,

CA, Cat#FB-02) and 1% Antibiotic-Antimycotic (Thermo Fisher Scientific,


and were added to new T150 flasks, where they were maintained for use in the

experiments. All cells were treated with inhibitors the day after they were plated.


SAH-SOS1A peptide (EMD Millipore, Temecula, CA, Cat#538092) was first mixed

in Tris buffer (50 mM Tris, 150 mM NaCl, pH 8.0) and the solution was further mixed with

a Q700 sonicator (QSONICA, Melville, NY). The SAH-SOS1A stock solution was further

diluted with the appropriate serum-free medium to concentrations of 0, 1.25, 2.5, 5, 10,

20, 40, and 80 µM. To inhibit mTOR, a 10 mM stock solution of sapanisertib (MedKoo

Biosciences, Research Triangle Park, NC) in DMSO was prepared and diluted to 0, 5, 10,

20, 40, 80, and 160 nM in medium.

Resazurin Cell Viability Assay

For assessment of cell proliferation and viability at different concentrations of

inhibitors, 104 cells per well were seeded into 96-well plates. Cells were incubated at 37ºC

for 48 hours in the appropriate media with varying inhibitor concentrations. The culture

supernatant was then removed and cells were incubated for 4 hours in Resazurin (R&D

Systems), a blue fluorescent indicator dye that is taken up by viable cells and

metabolically reduced to the highly fluorescent form, resorufin.45 Fluorescence was

measured with a SpectraMax® M5 Microplate Reader (Molecular Devices, San Jose, CA)

at excitation wavelengths of 544 nm and emission wavelengths of 590 nm.

Protein Assay Sample Preparation

To determine total protein concentrations of HPAF-II and BxPC-3 cell lysates after


Duplicate wells were dosed with each concentration of SAH-SOS1A the following day, and

plates were incubated at 37ºC for 48 hours. Cells were then washed with PBS, and RIPA

lysis buffer (MilliporeSigma, Burlington, MA, Cat#20-188) containing a protease inhibitor

cocktail (Roche, Cat#11697498001) and Phosphatase Inhibitor Cocktail A (Santa Cruz

Biotechnology, Cat#sc-45044) was added. After 15 minutes, cells were transferred to

tubes and incubated on ice for another 30 minutes. These were sonicated with

approximately 29 kJ and centrifuged at 10,000 x g for 10 minutes at 4ºC, and the PierceTM

bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Cat#23227) was used

to determine protein concentrations of the lysates. Absorbances of the standard and

sample solutions were measured at 562 nm using a SpectraMax® M5 to give the total

protein content in each sample.

To determine total protein concentrations of cell lysates after treatment with

sapanisertib, 3 x 105 cells per well were seeded into 6-well plates. The following day,

triplicate wells were dosed with each concentration of sapanisertib. After a 48-hour

incubation with the inhibitor at 37ºC, culture supernatant was removed. Each well was

scraped and cell contents were collected into separate 1.5 milliliter tubes with 1 mL of

phosphate-buffered saline (PBS). Cells were then disrupted by sonication with

approximately 25 kJ total. Protein concentrations of sonicated cell samples were

determined using the DC Protein Microplate Assay (Bio-Rad). Prepared bovine serum

albumin standards (Thermo Fisher Scientific) were used to create a standard curve. Five

microliters of standards or sonicated samples and 225 µL of assay reagents were added


temperature. Absorbances of the standard and sample solutions were measured at 750

nm using a SpectraMax® M5 to give the total protein content in each sample.

One-Stage Coagulation Assay

A metal ball was placed into each cuvette in the STart 4 Hemostasis Analyzer

(Diagnostica Stago, Parsippany, NJ). Fifty microliters of Pacific Hemostasis Coagulation

Control, Normal Level 1 plasma (Thermo Fisher Scientific, Cat#100595) in DEPC water

and 50 µL of each sonicated cell sample were added to every cuvette, and an

electromagnetic field was applied to oscillate the metal ball. Fifty microliters of 25 mM

CaCl2 in PBS were added to each cuvette to initiate clotting, and the times required for

coagulation (which stopped movement of the ball), were recorded as clotting times. TF

concentrations were determined using a standard curve produced with various

concentrations of Innovin (recombinant TF) (Siemens, Malvern, PA).

Statistical Analysis

Bars are presented as mean ± standard deviation. The Shapiro-Wilk test was used

to determine normality. For multiple comparisons, either the one-way ANOVA with

Dunnett’s multiple comparison test or the Kruskal-Wallis test with Dunn’s multiple

comparison test was used according to the normality of the data distributions. These

statistical analyses were performed with GraphPad PRISM version 8.4.0 (GraphPad



HPAF-II and BxPC-3 were incubated for 48 hours in various concentrations of each

inhibitor. We measured 4 main parameters: 1) cell viability through visual inspection, 2)

cell viability using the resazurin assay, 3) total protein concentration, and 4) cellular TF

activity. For all parameters, the responses of cells treated with the inhibitor were

compared to that of cells treated with vehicle. First, the presence of visible cell death

would indicate that the inhibitors are cytotoxic. Second, in the resazurin assay, a decrease

in fluorescence compared to untreated cells indicates that the inhibitor reduces cell

viability. Third, a decrease in total protein concentration of inhibitor-treated cells

compared to that of untreated cells indicates a cytotoxic effect on the cells. Fourth, a

decrease in TF activity with no effect on cell viability would indicate a selective inhibition

of TF expression whereas a decrease in both TF activity and cell viability would indicate

that the decrease in TF activity is secondary to cell death.

1) Effect of the KRAS inhibitor SAH-SOS1A on different parameters in HPAF-II and

BxPC-3 cells.

We treated cells with SAH-SOS1A to examine whether the KRAS pathway

contributes to TF gene expression in our PDAC cell lines. Substantial death of both

HPAF-II and BxPC-3 cells was visible in the 6-well plate cells treated with 40 µM and 80 µM

SAH-SOS1A, and almost all BxPC-3 cells observed at 80 µM were non-adherent. This

suggests that increasing concentrations of SAH-SOS1A were toxic to these cells. In

contrast to these visual observations, the resazurin assays showed only small, though

still significant, differences in fluorescence, with no concentration-dependence to these


TF activity in both cell lines with 40 µM and 80 µM SAH-SOS1A treatment (Figures

1C-1F). To independently analyze if the inhibitor was cytotoxic, cells were incubated with

SAH-SOS1A in 6-well plates and lysates were analyzed by western blotting

(Supplementary Materials, Methods). Phosphorylated ERK or GAPDH were greatly

reduced by western blotting in BxPC-3 lysates treated with 40 µM SAH-SOS1A (Figure


0 5 10 20 40 80 0.0

0.5 1.0 1.5

HPAF-II Protein

[SAH-SOS1A] (µM)

To ta l P ro te in C o n ce n tr at io n C

0 1.252.5 5 10 20 40 0

500 1000 1500 2000

[SAH-SOS1A] (µM)

Fluorescence (RFU)

HPAF-II Resazurin

** A

0 5 10 20 40 80 0.0 0.5 1.0 1.5 2.0 HPAF-II TF

[SAH-SOS1A] (µM)

Cellular TF



0 1.252.5 5 10 20 40 0 500 1000 1500 2000 BxPC-3 Resazurin

[SAH-SOS1A] (µM)

Fluorescence (RFU)

** * *


0 5 10 20 40 80 0.0

0.5 1.0 1.5

BxPc-3 Protein

[SAH-SOS1A] (µM)

To ta l P ro te in C o n ce n tr at io n D

0 5 10 20 40 80 0.0 0.5 1.0 1.5 2.0 BxPC-3 TF

[SAH-SOS1A] (µM)

Cellular TF



Figure 1. Effect of SAH-SOS1A on cell viability, protein levels, and cellular TF activity. (A, B) Fluorescence after

48-hour SAH-SOS1A treatment, n=8. Bars represent mean ± standard deviation, and asterisks label data with a significant difference compared to untreated: * p < 0.05 and ** p < 0.01. Total protein in HPAF-II (C) and BxPC-3 (D), and cellular TF activity of HPAF-II (E) and BxPC-3 (F) are shown. Values are shown relative to that of untreated (0 μM SAH-SOS1A) cells, n=2. Statistical analysis could not be performed with total protein or TF activity because only 2 independent experiments were performed.

2) Effect of the mTOR inhibitor sapanisertib on different parameters in HPAF-II and

BxPC-3 cells.

We next sought to determine if mTOR regulates TF expression in two PDAC cell

lines by examining the effect of sapanisertib treatment on the 4 parameters described

above. After cells were treated with sapanisertib, no HPAF-II cell death was visually

observed in the 96-well plate used for the resazurin assay, while some death of BxPC-3

cells was seen in the 96-well plate following treatment with 80 nM and 160 nM

sapanisertib. Interestingly, the resazurin assay showed a concentration-dependent

reduction in cell viability of HPAF-II and BxPC-3 cells treated with sapanisertib (Figures

2A and 2 B). The assay also produced a higher fluorescence signal for BxPC-3 cells than

for HPAF-II cells, which reflects the faster growth of BxPC-3. Sapanisertib also

concentration-dependently reduced total protein in HPAF-II and BxPC-3 cells (Figures 2C

and 2D). Interestingly, sapanisertib decreased TF expression in HPAF-II cells in a

concentration-dependent manner, but had no effect on TF activity in BxPC-3 cells


Figure 2. Effect of sapanisertib on cell viability, protein levels, and cellular TF activity. Fluorescence of HPAF-II (A) and

BxPC-3 (B) are shown, n=8. Total protein in HPAF-II (C) and BxPC-3 (D), and cellular TF activity of HPAF-II (E) and BxPC-3 (F) are shown. Bars represent mean ± standard deviation, n=3. Asterisks label data with a significant difference compared to untreated: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.


Our main goal was to examine the effect of KRAS and mTOR inhibitors on TF gene

expression in two human pancreatic cancer cell lines, HPAF-II and BxPC-3. We found 0 5 10 20 40 80 160

0.0 0.5 1.0 1.5 HPAF-II Protein [Sapanisertib] (nM) To ta l P ro te in C o n ce n tr at io n * * C

0 5 10 20 40 80 160 0.0 0.5 1.0 1.5 BxPc-3 Protein [Sapanisertib] (nM) To ta l P ro te in C o n ce n tr at io n * ** *** *** D

0 5 10 20 40 80 160 0.0 0.5 1.0 1.5 2.0 [Sapanisertib] (nM) Cellular TF Activity HPAF-II TF ** *** *** E

0 5 10 20 40 80 160 0.0 0.5 1.0 1.5 2.0 BxPC-3 TF [Sapanisertib] (nM) Cellular TF Activity F

0 5 10 20 40 80 160 0 2000 4000 6000 8000 [Sapanisertib] (nM) Fluorescence (RFU) HPAF-II Resazurin * ******* **** A


that both inhibitors induced cell death of the PDAC cell lines. Therefore, we were unable

to assess the roles of KRAS and mTOR in TF gene expression using these inhibitors

independently of the effect of cell death. Nevertheless, we made some interesting

observations during these studies.

First, treatment of HPAF-II and BxPC-3 with KRAS inhibitor SAH-SOS1A has a

cytotoxic effect on the cells. While SAH-SOS1A did yield a reduction in cellular TF activity

at the two highest concentrations, 40 µM and 80 µM, the concentration-dependent

decrease in total protein concentrations in both cell lines suggests that the lower TF

activity levels are largely attributed to cells being killed by the high inhibitor concentrations,

which we had visually observed. This was also reflected in the western blot of BxPC-3

lysates when we attempted to determine if SAH-SOS1A inhibited KRAS and did not

observe a signal for any of the probed targets in 40 µM-treated BxPC-3 (Figure S1B and

S1D). The absorbance of this lysate sample fell near the edge of the standard curve of

the protein assay, which makes the accuracy of protein concentration of the 40 µM

sample questionable.

It is unclear why the results of the resazurin viability assays (96-well plates) were

inconsistent with the decrease in cell viability as seen with our visual observation of cell

death (6-well plates), the total protein concentration, the TF activity, and the western blot.

It is possible that cells were more confluent in the 96-well plates and were therefore better

protected against SAH-SOS1A than the cells in the 6-well plates, which we used for the

protein assay, TF activity assay, and western blot. Another possible explanation is that

SAH-SOS1A may interfere with the resazurin assay. This can be confirmed in a future


Given these results, we cannot separate any reduction in KRAS-dependent TF

expression from that resulting from the cytotoxic effects of 48-hour SAH-SOS1A treatment.

The group that created SAH-SOS1A showed that the peptide inhibited KRAS kinase

activity through western blot detection of a variety of targets, including phosphorylated

ERK, but only treated their cells for 4 hours.26 However, our preliminary study with a

48-hour treatment of this inhibitor showed that SAH-SOS1A had no effect on HPAF-II

fluorescence in the resazurin assay, and it even increased fluorescence of BxPC-3

(Figures S2A and S2B). SAH-SOS1A also did not affect protein levels or cellular TF

activity in either cell line (Figures S2C and S2C). Therefore, this repeat experiment was

completed to verify those results. It is not known why our two experiments gave different

results. Conducting a time-course experiment with SAH-SOS1A, and with other KRAS

inhibitors as well, may help shed a light on a time- and concentration-dependent effect of

KRAS inhibition on TF gene expression in these cell lines.

The second focus of this study was the inhibition of mTOR by sapanisertib.

Sapanisertib decreased cell viability of both HPAF-II and BxPC-3, as measured by

reduced fluorescence in the resazurin assays and in decreased total protein

concentrations. Therefore, the reduced TF activity in HPAF-II is likely attributed to an

increase in cell death. We speculate that the resazurin and protein assays are more

sensitive than our visual inspection of cells in culture. Some cells may have appeared

adherent, but in response to toxicity may have exhibited morphological or metabolic

changes that we did not visually detect.

On the other hand, the cytotoxic effect measured in the BxPC-3 cell lysates is


sapanisertib. Our data is consistent with a previous study that showed that sapanisertib

treatment reduced cell survival of two other human PDAC cell lines, PANC-1 and MIA

PaCa-2.46 However, sapanisertib-treated BxPC-3 cells yielded unexpected outcomes that

are in contrast to the study of our collaborator, which showed TF suppression in

mTOR-inhibited BON.41 We observed a reduction in BxPC-3 cell viability but relatively constant

cellular TF activity with sapanisertib treatment, which suggests that the fewer surviving

cells demonstrated an enhanced level of TF activity equal to that of the more numerous

untreated cells. It is possible that sapanisertib actually decreases levels of the TF protein,

but increases its activity by some form of post-translational modification. The reason for

enhanced cellular TF activity in BxPC-3 at higher concentrations of sapanisertib requires

further investigation, such as through western blotting to measure the amount of TF


Enhanced BxPC-3 cellular TF activity with sapanisertib treatment despite cell

death may possibly also occur through mechanisms similar to those that occur with

inhibition of mTOR by rapamycin. It has been shown that rapamycin enhances thrombus

formation in vivo.47 Clinical case reports and reviews have also described thrombotic

events in patients after they received rapamycin either through drug-eluting coronary

stents,48,49 or after kidney50 and liver (with a higher, albeit insignificant, hepatic artery

thrombosis incidence)51 transplants.52 However, further clinical studies are required to

determine a causative effect of rapamycin on thrombosis. Guba et al.53 have also

described a relationship between rapamycin treatment and enhanced TF expression in


have shown this relationship in human aortic endothelial cells in vitro54 and A549

non-small cell lung cancer tumors in vivo.55 Rapamycin has also been found to stimulate TF

gene expression, although not activity, in human vascular smooth muscle cells.56

Guba et al.53 proposed that rapamycin both reduces signaling by the

PI3K/Akt/mTOR pathway, which they suggested is a negative regulator of TF expression,

and activates a TF-enhancing MAPK pathway especially in the presence of VEGF. Guha

and Mackman57 also found that the activated PI3/Akt pathway restricts the induction of

TF gene expression by lipopolysaccharide (LPS), and inhibition of the pathway therefore

increases LPS-induced TF gene expression. It is possible that sapanisertib had a similar

mTOR-downregulating and KRAS-upregulating effect on BxPC-3 in this study, and

therefore tipped the balance between KRAS and mTOR to induce TF gene expression.

To confirm the results of this present study, additional investigations with

rapamycin-mediated inhibition of mTOR inhibition should be performed. Furthermore, mTOR

inhibition should be verified with western blot detection of known mTOR downstream


It is possible that in BxPC-3 and HPAF-II, TF is more strongly regulated by

pathways not examined in this study. In future studies, an effective chemical inhibitor or

CRISPR-Cas9 could be used to knock down these genes in cells, such as BxPC-3 and/or

HPAF-II. Investigation of TF mRNA levels through RT-PCR could also provide information

about changes in gene expression. Such experiments would further elucidate the

relationship between the knocked down gene and TF gene expression in our search to



I wish to thank Dr. Nigel Mackman for always pushing me to become better at

science and allowing me to work in his lab, Dr. Yohei Hisada for mentoring me in the past

3 semesters, to Dr. Steven Grover for reading and editing this thesis, and to everyone in

the Mackman/Antoniak labs for their comments and constant support. I also would like

to thank Dr. Mark Peifer for being my Biology faculty sponsor, and Dr. Amy Maddox,

Mustafa Kemal Ozalp, and Julie Geyer for their support and help as well.


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Supplementary Materials


Figure S1. Western blots and densitometry analysis show levels of p-ERK and GAPDH in HPAF-II (A, C) and BxPC-3

(B, D). Cells were lysed after 48-hour treatment with SAH-SOS1A. GAPDH densitometry shows average GAPDH area under curve values relative to that of untreated cells (0 µM SAH-SOS1A), n=2.

0 5 10 20 40

0.0 0.5 1.0 1.5


[SAH-SOS1A] (µM)

Relative GAPDH


0 5 10 20 40

0.0 0.5 1.0 1.5


[SAH-SOS1A] (µM)

Relative GAPDH


40 20 10 5 0


37 kDa 45, 46 kDa


40 20 10 5 0


37 kDa 45, 46 kDa p-ERK



Figure S2. First experiment with SAH-SOS1A. Fluorescence of HPAF-II (A) and BxPC-3 (B) after 48-hour incubation with different concentrations of SAH-SOS1A at 37º, n=8. Protein concentrations of HPAF-II (C) and BxPC-3 (D), and cellular TF activity of HPAF-II (E) and BxPC-3 (F). Bars for total protein and TF activity show mean ± standard deviation, n=3. Asterisks label data with a significant difference compared to untreated: * p < 0.05, ** p < 0.01, and *** p < 0.001.

0 500 1000 1500 2000 2500 3000

0 1.25 2.5 5 10 20 40

Fl uor es cenc e (R FU ) [SAH-SOS1A] (µM) BxPC-3 Resazurin


*** ** ** * * 0 20 40 60 80 100 120 140 160 180 200

0 1.25 2.5 5 10 20 40

Tot al P rot ei n Co nc en tr at io n (µ g/ m L) [SAH-SOS1A] (µM) HPAF-II Protein


0 20 40 60 80 100 120 140 160 180 200

0 1.25 2.5 5 10 20 40

Tot al P rot ei n Co nc en tr at io n (µ g/ m L) [SAH-SOS1A] (µM) BxPC-3 Protein


0 1 2 3 4 5 6 7 8 9 10 11

0 1.25 2.5 5 10 20 40

Ce llu la r T F Ac tiv ity (n g/m L) [SAH-SOS1A] (µM) HPAF-II TF


0 500 1000 1500 2000 2500 3000

0 1.25 2.5 5 10 20 40

Fl uor es cenc e (R FU ) [SAH-SOS1A] (µM) HPAF-II Resazurin


0 1 2 3 4 5 6 7 8 9 10 11

0 1.25 2.5 5 10 20 40



SDS-PAGE of Cell Lysates Treated with SAH-SOS1A

For electrophoresis, SDS sample buffer (Invitrogen, Carlsbad, CA, Cat#NP0007)

with 5%β-mercaptoethanol was added to cell lysates, which were heated to 95ºC for 10

minutes. Samples (3.1 µg protein/well for HPAF-II; 1.2 µg protein/well for BxPC-3) were

loaded onto 4-20% gradient Mini-PROTEAN® TGXTM gels (BioRad Laboratories,

Hercules, CA, Cat#456-1093) with Precision Plus ProteinTM KaleidoscopeTM Prestained

Protein Standards (BioRad Laboratories, Hercules CA, Cat#161-0375) as a marker for

molecular weight. Electrophoresis in Tris/Glycine/SDS buffer (BioRad Laboratories,

Cat#161-0732) was used to separate proteins.

Western Blotting

Proteins were transferred to polyvinylidene difluoride membranes (MilliporeSigma,

Cat#IPFL00010) and blocked with protein-free blocking buffer (Thermo Fisher Scientific

Cat#23227). Membranes were probed using Phospho-p44/42 (ERK1/2) (Thr202/Tyr204)

(D13.14.4E) XP® Rabbit mAb (Cell Signaling Technologies, Danvers, MA, Cat#4370),

p44/42 (ERK1/2) (137F5) Rabbit mAb (Cell Signaling Technologies, Cat#4695) and goat

anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polyclonal antibody (Abcam,

Cambridge, MA, USA, ab9483), which were diluted 1:2000, 1:1000, and 1:200,

respectively, in blocking buffer. Blots were then washed with Tris-buffered saline with

0.1% Tween20 (TBS-T) and incubated with IR Dye® 680RD goat anti-rabbit IgG (LI-COR,

Lincoln, NE, Cat#926-68071) or IRDye® 800CW Donkey anti-Goat IgG (LI-COR,

Cat#926-32214), each diluted to 1:10k in blocking buffer. Blots were developed with an





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