Fig. 4.

HACE1 controls melanoma cell adhesion/migration through the regulation of fibronectin secretion. a Circular network identified by Ingenuity Pathway Analysis (Qiagen®) showing the relationship between fibronectin (FN) and genes that are downregulated by HACE1 silencing. b A375 cells were transfected with control (siCtl) or HACE1 (siH2) siRNA and analysed via western blotting of cell lysate (CL) using antibodies against HACE1, ITGβ1, ITGαV, FN, and actin (loading control). Conditioned medium (CM) was probed with an antibody against FN and vitronectin (VN). c Three different melanoma cell cultures were transfected with control (siCtl) or HACE1 (siH1) siRNA. The CM was analysed by western blot analysis using antibodies against FN or VN. HACE1 suppression was verified by western blot analysis of cell lysate proteins with HACE1 antibodies. d C-13.08 cells were transfected with control (siCtl) or HACE1 (siH1) siRNA or cultured in CM from cells treated with control (CM-siCtl) or HACE1 (CM-siH1) siRNA. Cells were then subjected to Boyden chamber migration assays. e The protein extracts from cells treated as described in D were analysed by western blotting with antibodies against HACE1 or ITGαV. HSP90 was used as a loading control. f 501MEL cells were seeded on plates coated with the following treatments: CM from 501MEL cells treated with siCtl (CM-siCtl) or siHACE1 (CM-siH1); fibronectin-free (immuno-depleted) CM from control 501MEL cells (CM-siCtl w/o FN); CM from HACE1-suppressed 501MEL cells supplemented with 0.5 µg/ml recombinant fibronectin (CM-siH1 + FN). g Quantification of three separate experiments as described in F. The results represent the mean ± SD of the percent of cell adhesion in the control condition. h Western blot analysis of secreted fibronectin after immuno-depletion with control (Ctl) or FN antibodies