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. 2017 Oct 16;114(44):E9271–E9279. doi: 10.1073/pnas.1703921114

Fig. 3.

Fig. 3.

Targeting autophagy induces the up-regulation of CCL5 mRNA by increasing the phosphorylation of c-Jun. (A, Left) Expression of Beclin1, total c-Jun, phosphorylated c-Jun on serine 63 (pS63), and serine 73 (pS73) proteins in cell extracts from Ctrl and BECN1− B16-F10 cells. Actin was used as loading control. (Right) Expression of Beclin1, phosphorylated c-Jun on serine 63 (pS63), and serine 73 (pS73) proteins in three regions (#1, #2, #3) of Ctrl and BECN1− B16-F10 tumor extracts. Actin was used as loading control. (B) Expression of Beclin1, total and phosphorylated SEK/MKK4 on Ser-80, Thr-261, and Ser-257, and phosphorylated JNK on Thr-185/Tyr-183 in Ctrl and BECN1− B16-F10 melanoma cells. (C, Upper) Expression of Beclin1, phosphorylated JNK on Thr-185/Tyr-183, and phosphorylated c-Jun on serine 63 (pS63) and serine 73 (pS73) proteins in Ctrl and BECN1− cells untreated (−) or treated (+) with JNK inhibitor SP600125. Actin was used as loading control. (Lower) The expression of CCL5 mRNA in cells described in the Upper panel. Data are reported as fold-change (FC) compared with the Ctrl. *P < 0.05 (two-tailed Student's t test). (D, Upper) BECN1− B16-F10 melanoma cells were transfected with control (Ctrl) or JNK1/2 (JNK) siRNA. The expressions of JNK, Ser-63 (pS63), and Ser-73 (pS73) phosphorylated c-Jun were assessed 72 h after transfection. Vinculin was used as loading control. (Lower) The expression of CCL5 mRNA by real-time RT-PCR in cells described in A. Data represent the average ± SD of three independent experiments. ***P < 0.005 (two tailed Student’s t test). (E) PP2A phosphatase activity assay in Ctrl and BECN1− B16-F10 tumor cells. Data reported as picomoles of free phosphate released following incubation of PP2Ac immunoprecipitated from each cell with Threonine phosphopeptide. The result shown is the average ± SEM of five independent experiments performed in duplicate. *P < 0.05 (two-tailed Student’s t test). (F, Left) Expression of PP2A subunit A, total and phosphorylated c-Jun on serine 63 (pS63) and serine 73 (pS73) and total and phosphorylated JNK on Thr-185/Tyr-183 proteins in Ctrl B16-F10 cells untransfected (−) or transfected with PP2A siRNA targeting A subunit. Actin was used as loading control. (Right) Expression of CCL5 mRNA in the cells described in the Left panel. Data are reported as fold-change (FC) compared with the Ctrl. Results represent the average ± SEM of three independent experiments. **P < 0.01 (two-tailed Student’s t test). (G, Upper) Expression of phosphorylated PP2A on Tyr-307 PP2A and phosphorylated c-Jun on serine 63 (pS63) in Ctrl B16-F10 cells untreated (−) or treated (+) with okadaic acid (OA). (Lower) Quantification of CCL5 secreted by cells described in the Upper panel as determined by ELISA.