Figure 5
Resistin stimulates invasion of breast cancer cells through ERM.
MDA-MB-231 cells were treated with (A) different doses of resistin and cultured for 1 h, or (B) treated with resistin (25 ng/ml) for the indicated times. Cell lysates (30 μg) were analyzed by western blots using antibody against phospho-ERM; ERM was used as the control. (C) Cells were immunoprecipitated with anti-PKCα antibody, followed by western blots with anti-ERM and PKCα antibodies. (D) Cells were treated with resistin for the indicated times and the lysates were immunoprecipitated with anti-PKCα antibody, followed by western blots with anti-ERM and PKCα antibodies. (E) Cells were treated with Gö 6976. Cell lysates (30 μg) were analyzed by western blots using antibodies against phospho-ERM and phospho-PKCα; ERM and PKCα were used as controls. (F) Cells were pretreated with Gö 6976 (10 μM) and incubated with resistin for 60 min. Cell lysates were analyzed by western blots using antibodies against phospho-ERM and phospho-PKCα; ERM and PKCα were used as controls. (G) HA-tagged PKCα (KD)-expressing cells were treated with resistin for 1 h. Cell lysates were analyzed by western blots using anti-phospho-ERM and HA antibodies; ERM and β-actin were used as controls. (H) Cells were transfected with non-target or ezrin siRNA for 48 h. A cell-free space was created by scraping through the monolayer. Migration was induced by 25 ng/ml resistin for 16 h. (I) Effect of ezrin suppression on cell invasion. Cells were transfected with non-target or ezrin siRNA for 48 h. Cells were subjected to an invasion assay and stained and randomly chosen fields were imaged at 100× magnification. The number of cells invading the lower surface was counted. (J) Representative images (ezrin and objective image) of ezrin-GFP-expressing cells treated with resistin for 1 h. Scale bars, 10 μm. (K) Representative images (ezrin-GFP, PKCα and bright field) of cells treated with resistin for 1 h. Scale bars, 10 μm. *P < 0.05 vs. untreated condition.
