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. 2017 Apr 21;9(4):37. doi: 10.3390/cancers9040037

Figure 4.

Figure 4

PDK1 is activated by Timp1, along with melanoma progression, and contributes to cell survival. (A) PDK1 activation was examined in cell lines 4C, 4C11−, and 4C11+ and their Timp1-silenced clones. Total PDK1 and β-actin were used as loading controls. WT: non-treated cells; non-target: control non-target shRNA; shTimp1#2: clone 2 silenced for Timp1; shTimp1#3: clone 3 silenced for Timp1; (B) cell lines treated with LY294002 (PI3K inhibitor), GSK2334470 (PDK1 inhibitor), and Bisindolylmaleimide I (PKC inhibitor) were analyzed for PDK1 activation by Western blotting. Total PDK1, β-actin (Actin), and GAPDH were used as loading controls; (C) 4C11+ metastatic melanoma cells treated or not with LY294002 (PI3K inhibitor) were analyzed for AKT activation by Western blotting. β-actin was used as a loading control; (D) cell lines non-treated (NT) or treated with GSK2334470 (PDK1 inhibitor) were evaluated for anoikis resistance after maintained in suspension for 96 h; (E,F) colony formation capacity was determined in cell lines non-treated (NT) or treated with GSK2334470 (PDK1 inhibitor); and (G) cell lines corresponding to both early (4C and 4C11−) and late (4C11+) stages of melanoma progression confer cell survival via Timp1 by activating PDK1 pathway. In metastatic cell line (4C11+), AKT also contributes to anoikis resistance. * p < 0.05, ** p < 0.01, *** p < 0.001.