(C) Results of a GST pull\down assay using GST\PAK1\CRIB\coated Sepharose beads showing that Sos1 knockdown completely inhibits EGF\induced Rac1 activation compared with cells transfected with non\targeting (Ctl) siRNA in Capan1, BxPC3, and Panc0403 cells. a ratio of Ctl. PATH-243-37-s003.tif (583K) GUID:?29EC60B2-B504-48AB-A826-70A7D4F45A3F Figure S2. v6 inhibition does not affect PDAC cell migration towards BSA. (A) Western blot showing v expression in PDAC cell lines. Equal loading was confirmed by HSC70. (B) 50 000 PDAC cells were pretreated with 10 g/ml of the v6 blocking antibody 63G9 for 30 min before plating them into the top well of bovine serum albumin\coated Transwell? migration inserts. The number of cells that migrated to the bottom wells was counted after an overnight incubation and no change in the number of migrating cells was detected upon pretreatment with the blocking antibody. Note the low number of migrating cells. Diagram represents Mc-Val-Cit-PABC-PNP the mean number of migrating cells per well SD; n = 3. PATH-243-37-s010.tif (116K) GUID:?19D02FFD-D148-4369-B46C-9399F8446FEC Figure S3. v6\positive PDAC cells activate TGF\1. PDAC cells (120 000) were plated on top of MLEC cells and TGF\1 activation was measured after an overnight incubation. The v6\positive Capan1, BxPC3, and Panc0403 cancer cells induced significant activation of TGF\1, while v6\negative SW1990 and SU86.86 cells did not. Diagram represents relative light units expressed as a % of Ctl SD; n = 6; *p 0.05; ***p 0.001; ****p 0.001; ns = non\significant. PATH-243-37-s007.tif (236K) GUID:?A03C0715-95E6-42DB-B599-E24490716E0F Figure S4. Eps8 knockdown using four siRNA sequences inhibits PDAC cell migration and induces TGF\1 activation. (A) Transwell? migration of BxPC3 Mc-Val-Cit-PABC-PNP cells towards LAP was significantly inhibited by transfection with the Eps8 siRNA sequence used throughout the study (Eps8) and three alternative siRNA sequences targeting Eps8 (?1\2\3). Diagram represents the mean number of migrating cells per well expressed as a % of Ctl (BSA) SD; n = 3; *p 0.05. (B) Eps8 knockdown using four individual siRNA sequences induces activation of TGF\1 in BxPC3 cells measured by an MLEC TGF\ activation assay. Diagram represents the mean relative light units expressed as a % of Ctl SD; n = 6; **p 0.01; ***p 0.001; ****p 0.0001. Western blots confirm down\regulation of Eps8 using RNA interference. Equal loading was confirmed by HSC70. Numbers below the blots indicate the densitometry values measured using ImageJ normalized to HSC70 and expressed as a ratio to Ctl. PATH-243-37-s004.tif (173K) GUID:?3E94EE81-70BE-4AA8-AEAE-120506DF5F26 Figure S5. Eps8 overexpression increases cell motility while it inhibits TGF\ activation. (A) Capan1, BxPC3, and Panc0403 cells were transfected with empty vector (EV) or Eps8CEGFP 24 h before plating them into a Transwell? migration Mc-Val-Cit-PABC-PNP assay. Eps8 overexpression in all three cell lines significantly increased cell migration towards the v6 ligand, LAP. Diagram represents the mean number of migrating cells per well expressed as a % of Ctl (BSA) SD (Capan1/Panc0403 plotted on left, BxPC3 plotted on right Y\axis); n = 3; *p 0.05; ***p 0.001. (B) Capan1, BxPC3, and Panc0403 cells were transfected with empty vector (EV) or Eps8\EGFP 24 h before plating them on top of MLEC cells. Eps8 overexpression significantly inhibited TGF\ activation in all three cell lines. Diagram Mc-Val-Cit-PABC-PNP represents the mean relative light units expressed as a % of MLECs SD (Capan1/Panc0403 plotted on left, BxPC3 plotted on right Y\axis); n = 6; **p 0.01; ****p 0.0001. (C) Eps8CEGFP expression was confirmed by western blotting. HSC70 was used as a loading control. PATH-243-37-s014.tif (357K) GUID:?0F57DEAD-23D5-419A-B501-6A0A840C1881 Figure S6. Eps8 does not affect the cell surface levels of 6 integrin. Cells were transfected with non\targeting (Ctl) or Eps8\targeting siRNA, and the cell surface levels Mc-Val-Cit-PABC-PNP of total (A) or active (B) 6 integrin were measured by FACS analysis 48 h post\transfection using either anti\6 (620 W) (A) or anti\active 6 (6.2G2) antibodies (B). Diagrams represent the mean fluorescence intensity expressed as a % of Ctl SD; n = 3; ns = non\significant. (C) Cells were transfected with either non\targeting (Ctl) or Eps8\targeting siRNA, and cell adhesion on LAP was measured 48 h post\transfection. Eps8 down\regulation had no effect on v6\specific adhesion of PDAC cells. Diagrams represent the absorbance at 540 nm expressed as a % of Ctl (BSA) SD; n = 4; ns = non\significant. Western blots confirmed down\regulation of Eps8 following siRNA transfection. Equal loading was confirmed by HSC70. Numbers below the blots indicate the densitometry values measured using ImageJ normalized to HSC70 and expressed as a ratio to Ctl. PATH-243-37-s006.tif (291K) GUID:?9A9CB58E-098B-4C9C-8419-5F3244A84FA9 Figure S7. EGF stimulation potentiates v6 signalling and function. (A) Western blot showing expression of EGFR in the v6\positive Capan1, BxPC3, and Panc0403 cancer cells. The SCC25 oral squamous cell carcinoma cell line was used as a positive control. Equal Mouse monoclonal to SARS-E2 loading was confirmed by HSC70..