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

Figure 5.

Figure 5

Timp1 modulates the PKC activation via PDK1 in the early stages of melanoma. (A) PKC activation was analyzed by Western blotting in the melan-a (ma), 4C, 4C11−, and 4C11+ cell lines. Total PKC and β-actin (Actin) were used as loading controls. (B) PKC activation was analyzed by Western blotting in cell lines silenced for Timp1. Total PKC and β-actin (Actin) were used as loading controls. WT: non-treated cell lines; non-target: control non-target shRNA; shTimp1#2: clone 2 silenced for Timp1; and shTimp1#3: clone 3 silenced for Timp1. (C) PKC activation was determined in cell lines (4C and 4C11−) treated with LY294002 (PI3K inhibitor) or GSK2334470 (PDK1 inhibitor). The control was subjected to the same conditions without any treatment (non-treated). Total PKC and β-actin (Actin) were used as loading controls. (D) Anoikis resistance was analyzed in pre-malignant 4C melanocytes and non-metastatic melanoma 4C11- cell lines, non-treated and treated with Bisindolylmaleimide I (PKC inhibitor). (E,F) Colony formation was evaluated in 4C and 4C11- cell lines, non-treated and treated with Bisindolylmaleimide I (PKC inhibitor). (G) Cell survival is favored by Timp1 in early stages of melanoma progression by activation of the PDK1/PKC pathway. In late stages (metastasis), both PDK1 activated by Timp1 and AKT independently of Timp1 contribute to cell survival. melan-a: melanocytes; 4C: pre-malignant melanocytes; 4C11−: non-metastatic melanoma cell line; 4C11+: metastatic melanoma cell line; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.