Figure 2. Collagen I-induced stabilization of Snail1 protein requires DDR2.

(a) HEK293 cells transfected with indicated plasmids (CTL – empty vector) were added to plates coated with collagen I (2 mg/ml) for indicated times (hours). Western blots were performed. (b) Human MDA-MB-231 cells were added to plates coated with ECM proteins for 8 hours: FN – fibronectin; Gel – gelatin. DDR2 was immunoprecipitated and bound products Western blotted with pTyr or DDR2 antibodies (upper panels). Western blots of cell extracts (lower panels). In the last two lanes cells were depleted of DDR2 with shRNAi. (c) MDA-MB-231 cells were untreated (CTL), pretreated with mouse IgG or neutralizing antibody against β1-integrin (MAb13) for 2 hours, added to collagen I coated (+) or uncoated plates (−) for 15 minutes or 8 hours. Western blots were performed. (d) MDA-MB-231 cells were treated with DMSO or SB431542 (10 µM), an inhibitor of TGFβ signaling, before addition to plates coated with collagen I (+) or uncoated (−) for 8 hours. Western blots were performed. (e) MCF-10A cells infected with DDR2 or scrambled control (SCR) shRNAi lentiviruses were treated with TGFβ (2 ng/ml) continuously for 4 days to induce EMT. Western blots were performed on cell extracts each day for 4 days of TGFβ treatment. (f) MCF-10A cells were infected with empty (CTL), Snail1-Flag, or myc-DDR2 retroviruses and cultured without TGFβ for 4 days. Phase images of cells on day 4 (left). Scale bar 100 µm. Western blots of day 4 cell extracts with the indicated antibodies (right).