Wp1066

Antibodies specific for CD147 (sc-9754), α-tubulin (sc-8035), WAVE2 (sc-10392), donkey anti-goat IgG-FITC (sc-2024) and FAK Inhibitor 14 (Y-15) (sc-203950) were purchased from Santa Cruz (Dallas, Texas); phospho-FAK (Tyr397) (3283), phospho-Src (Tyr416) (2013), phospho-myosin light chain 2 (Thr18/Ser19) (3674), Src (2109), and myosin light chain 2 (3672) antibodies were purchased from Cell Signaling Technology (Boston, MA); DOCK8 (11622-1-AP) and STAT3 (60199-1-Ig) antibodies were obtained from Proteintech (Wuhan, China); phospho-STAT3 (Tyr705) (ab76135) antibody was obtained from Abcam (Cambridge, UK); phospho-Src (Tyr527) (orb14869) antibody was obtained from Biorbyt LLC (San Francisco, CA); and Alexa 594-conjugated goat anti-mouse IgG and Alexa Fluor 488 phalloidin were purchased from Invitrogen (Carlsbad, CA). The Src kinase inhibitor Src I-1, FAK inhibitor PF573,228 (Sigma, St. Louis, MO) and STAT3 inhibitor WP1066 (Merck Millipore, Darmstadt, GER) were also used. Src I-1 was used at 300 nM, PF573,228 at 300 nM, Y-15 at 10 μM and WP1066 at 5 μM. The Cell Proliferation Reagent WST-1 was purchased from Roche (Mannheim, GER).

The STAT3 inhibitor WP1066 used in this study was purchased from Selleckchem. Cells were seeded in 24‐well plates (5 × 10⁴ cells/well) and 12‐well plates (1 × 10⁵ cells/well) and cultured overnight. To assess the effects of the inhibitor, cells were starved overnight with serum‐free medium and then incubated with starvation medium alone, 5 ng/mL of human recombinant TGFβ1 (Roche) with and without 5 µmol/L and 10 µmol/L WP1066 for 24 hours. Cells (24‐well plates) were then fixed with chilled acetone: methanol (1:1), dried and stained for different markers (collagen‐I, α‐SMA, and vimentin) (antibodies are summarized in Table S1). In addition, cells (12‐well plates) were lysed with RNA lysis buffer to perform quantitative real‐time PCR analyses or protein lysis buffer for western blot analyses.