1. A B HPAF-II CFPAC-1 cells 0 200 400 600 800 Drug concentration, µM 50
Supplemental Figure 1
Mattheolabakis et al A
HPAF-II
CFPAC-1 cells
Drug concentration, µM 50
100
Cell growth, % control
Drug concentration, µM 50
100
Cell growth, % control B
MIA PaCa-2
Panc-1
PA, µM
ASA, µM
400
Supplemental Figure 1. Phospho-aspirin inhibits the growth of human PC cells. A: PA inhibits PC cell growth in a concentration- dependent manner. Cell growth was determined in human HPAF-II CFPAC-1 cells after treatment with escalating concentrations of PA or aspirin (ASA) for 24 h. Results are expressed as % control. B: PA inhibits colony formation in a concentration-dependent manner in MIA PaCa-2 and Panc-1 cells. Aspirin (ASA), 400 µM had no effect. Images of each condition at the end of the 7 day-period are shown.

2. Supplemental Figure 2
Mattheolabakis et al A
Annexin V (+) cells,
Fold increase over control * B
Time, h
PA, 1.5xIC
Caspase -3
Survivin
β-tubilin
Supplemental Figure 2. Phospho-aspirin induces apoptosis in human PC cells. A: Human Panc-1, MIA PaCa-2 or HPAF-II PC cells treated with PA 1.5xIC50 for 24 h were stained with Annexin V/propidium iodide, and the percentage of apoptotic cells was determined by flow cytometry. Results are expressed as mean ± SEM. *Significant different compared to control (p<0.01). B: PA reduces full length caspase-3 and survivin levels. Panc-1 cells were treated with PA, for various periods of time, as shown. Immunoblots for full length casepase-3, survivin. β-tubulin = loading control.

3. Supplemental Figure 3
Mattheolabakis et al B A
Ac-p53 (K382)
Ac-p53 (K379)
p53
PA, xIC50
Vehicle PA
Ac-p53 (K382)
Vehicle PA
% Ac-p53 (K382) (+) cells 10 20 30 * C
Time, h
p21
β-actin
PA, 1.5xIC
Supplemental Figure 3. Phospho-aspirin induces p53 acetylation in human PC cells and xenografts. A: Effect of PA on p53 acetylation (Ac-p53) and the level of p53 in MIA PaCa-2 cells treated with PA for 2 h. B: Representative images and the quantification of Ac-p53 (K382) expression in tumor sections. The percentage of Ac-p53 positive cells in vehicle or PA-treated Panc-1 xenografts was determined by immunohistochemical staining. All values are mean ± SEM; *p<0.05, compared to vehicle control. C: Effect of PA on p21 expression in Panc-1 cells. Panc-1 cells were treated with PA, for various periods of time, as shown. Immunoblot for p21 is shown. β-actin = loading control.

4. PA, µM ASA, µM A23187 - + + + + + 400 600 400 600 10 20 30 PGE2, pg/ml
Supplemental Figure 4
Mattheolabakis et al
PA, µM
ASA, µM A 10 20 30
PGE2, pg/ml *
Supplemental Figure 4: PA does not inhibit PGE2 levels in PC cells. Panc-1 cells were pretreated with various concentrations of PA or ASA for 30 min, followed by treatment with the calcium ionophore A µM for 3 h. PGE2 levels in the culture medium were measured by enzyme- linked immunosorbent assay.

5. MIA PaCa-2 AsPC-1 PANC-1 HPAF-II p-EGFR (Y1045) p-EGFR (Y1068)
Supplemental Figure 5
Mattheolabakis et al
p-EGFR (Y992)
p-EGFR (Y1045)
p-EGFR (Y1068)
β-actin
AsPC-1
PANC-1
MIA PaCa-2
HPAF-II
EGFR
Supplemental Figure 5. Expression levels of phospho-EGFR (p-EGFR) in multiple human PC cells. Levels of p-EGFR expression levels at various sites was performed by WB .

6. B A C * MIA PaCa-2 cells AsPC-1 cells IF intensity values PA Control
Supplemental Figure 6
Mattheolabakis et al A
MIA PaCa-2 cells B
AsPC-1 cells PA
Control
EGF
p-EGFR
Merge DAPI PA
Control
EGF
p-EGFR
Merge DAPI C
IF intensity values *
Supplemental Figure 6. Phospho-aspirin inhibits EGFR phosphorylation in PC cells. A-B: Phospho-aspirin (PA) inhibits EGFR phosphorylation in MIA PaCa-2 cells and AsPC-1 cells. Immunofluorescence staining for p-EGFR was performed in MIA PaCa-2 or AsPC-1 cells treated or untreatead with PA (1xIC50) for 4 h. EGF (10 ng/ml) stimulation for 15 min was used as positive control. Representative images in each group from three independent experiments are shown (original magnification, x20). The levels of p-EGFR and DAPI was observed using a Nikon ECLIPSF 90i microscope. C: Immunofluorencesce intensity in each group were quantified and results expressed as mean ± SEM. *Significant different compared to control (p<0.01).

7. 4000 8000 12000 Amylase, U/L CTR CTR PA CTR CTR PA Cerulein 1000 2000
Supplemental Figure 7
Mattheolabakis et al
4000
8000
12000
Amylase, U/L
CTR CTR PA CTR CTR PA
Cerulein
3d following last cerulein injection
1h following last cerulein injection
1000
2000
3000
Lipase, U/L
CTR CTR PA CTR CTR PA
Cerulein
3d following last cerulein injection
1h following last cerulein injection
Supplemental Figure 7. Serum amylase and lipase levels. Six week old KrasG12D mice (C57Bl/6J background) were injected intraperitoneally with saline or cerulein (250 μg/kg hourly, once per day for three consecutive days). Cerulein-injected mice were divided into vehicle control (CTR) or PA-treated groups (n=5/group). PA 100 mg/kg was given orally once a day, starting on the day of the first cerulein injection (See Fig 4A). One hour or three days after the last cerulein injection, mice were euthanized and serum was collected. Amylase and lipase levels were determined by a commercial laboratory (Antech Diagnostics).

8. Supplemental Figure 8
Mattheolabakis et al
day day 5
Supplemental Figure 8. Representative images of acinar cells explants at the time of plating (day 0) and of the resulting metaplasia (day 5) after five days of incubation. (x40)