Fig. 5

KRT16 stabilizes β5-integrin and c-Met to transmit downstream signaling through Src/STAT3 signaling pathway. a Analysis of integrin isoforms in KRT16-siRNA–transfected OC-3-IV and C9-IV3 cells. b Treatment with 10 μM MG132 for 8 h could not prevent the loss of β5-integrin (ITGB5) in KRT16-depleted cells. Treatment with 100 nM bafilomycin A (B.A.) for 24 h prevented ITGB5 degradation in KRT16-depleted cells. c NC-siRNA and β5-integrin-silenced OC-3-IV cells were serum starved for 18 h O/N, treated with 20 ng/mL HGF for 30 min, and subjected to immunoblotting. Depletion of KRT16 depletion enhanced degradation of c-Met in OC-3-IV cells. Knockdown of KRT16 decreased c-Met protein, which was blocked by B.A. rather than MG132. d Left, OC-3-IV cells were transfected with the indicated plasmids for 8 h, serum starved for 24 h, and then were treated with 50 ng/mL HGF for the indicated times. Right, the p-c-Met and c-Met levels were shown and quantified. e Localization of KRT16, c-Met, and β5-integrin was analyzed in OC-3-IV cells through confocal microscopy. Panel a, IF staining under confocal microscopy of KRT16 (red), β5-integrin (green), and nuclei (DAPI, blue) after adhesion of OC-3-IV cells to Poly-L-Lysine; panel b, IF staining under confocal microscopy of KRT16 (red), c-Met (green), and nuclei (DAPI, blue); panel c, IF staining under confocal microscopy of β5-integrin (red), c-Met (green), and nuclei (DAPI, blue). f Cell lysates were blotted directly or subjected to IP with the indicated antibodies followed by blotting with the indicated antibodies. IgG served as the negative control. g Effect of KRT16 knockdown on c-Met signaling-related molecules in OC-3-IV and C9-IV3 cells. All the in vitro experiments were performed in triplicates and repeated three times. The actin was used as an internal control