Fig. 3.

FAK activation controls cell growth and migration in USC cell lines. (A) Representative western blot of p‐FAK‐Y³⁹⁷, total FAK, and pPAX‐Y¹¹⁸ levels in ARK‐1 cells treated with 0.6 nm, 500 nm, 1 μm, and 5 μm of defactinib for 1 h (n = 3 independent experiments). β‐actin expression is used as a loading control. (B) Representative phase contrast images of ARK‐1 3D cells treated with 10 μm PF‐573228 or 5 μm defactinib for 48 h (n = 3 independent experiments). Magnification 10×. Scale bar: 100 μm. Representative western blot of p‐FAK‐Y³⁹⁷, total FAK, and cyclin D1 levels in ARK‐1 cells treated with 5 μm of defactinib for 0, 6, 18, and 24 h. (n = 3 independent experiments). β‐actin expression is used as a loading control. (C) Quantification of glandular perimeter (μm) in ARK‐1 3D cells treated with 10 μm PF‐573228 or 5 μm defactinib for 48 h. Percentage of positive BrdU ARK‐1 cells in a 3D sphere experiment where cells were treated with 0.6 nm, 500 nm, 1 μm, and 5 μm of defactinib for 48 h. Values are means±SD (n = 3 independent experiments); One‐way ANOVA test was used followed by Tukey post hoc test (***P < 0.001). (D) Quantitative analysis of velocity plot (μm·min⁻¹) and accumulated distance (μm) of ARK‐1 cells, treated with either 10 μm PF‐573228 or 5 μm Defactinib. Values are means ± SD (n = 3 independent experiments); One‐way ANOVA test was used followed by Tukey post hoc test (***P < 0.001). (E) Western blot of p‐FAK‐Y³⁹⁷, total FAK and cyclin D1 levels in ARK‐1 scramble and ARK‐1 FAK shRNA cells (n = 3 independent experiments). β‐actin expression is used as a loading control. (F) Representative images and quantification of glandular perimeter (μm) in scramble and FAK shRNA ARK1‐3D spheroids (n = 3 independent experiments); Student's t‐test (***P < 0.001). Scale bar: 100 μm. (G) Quantitative analysis of velocity plot (μm·min⁻¹) and accumulated distance (μm) of ARK‐1 scramble vs. FAK shRNA cells. Values are means±SD (n = 3 independent experiments); Student's t‐test (***P < 0.001).