Fig. 9

FPS-ZM1 interferes with Ins-induced stimulatory pathways in CAFs and 4T1 cells. Representative immunoblots showing the phosphorylation of IR (Y1135/1136) (A), IRS1 (Y612) (B), and AKT (S473) (C) in CAFs treated with Ins (20 nM, 15 min) alone and in combination with FPS-ZM1 (10 μM, 24 h). Treatment with FPS-ZM1 (10 μM, 24 h) abrogates the up-regulation of CYR61 protein expression D in CAFs stimulated with Ins (20 nM, 4 h). Total proteins and β-actin serve as loading control. Wound healing assay in CAFs scratched and treated with Ins (20 nM) alone and in combination with FPS-ZM1 (10 μM) in serum free medium. Images are acquired at 0 and 16 h after scratching, as indicated (E). Side panel shows the quantification of cell migration expressed as % of wound closure. Representative immunoblots showing the phosphorylation of IR (Y1135/1136) (F), IRS1 (Y612) (G), and AKT (S473) (H) in 4T1 cells treated with Ins (20 nM, 15 min) alone and in combination with FPS-ZM1 (10 μM, 24 h). Treatment with FPS-ZM1 (10 μM, 24 h) abrogates the up-regulation of CD1 protein expression in 4T1 cells stimulated with Ins (20 nM, 4 h) (I). Total proteins and β-actin serve as loading control. 4T1 cells were seeded in 96-well plates and treated with either vehicle (−) or Ins (20 nM), alone and in combination with FPS-ZM1 (10 μM) for 72 h before evaluation of cell growth by SRB assay (J). Data shown are mean ± SEM of at least three independent experiments performed in duplicate. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001