Fig. 6. Silencing ANO1 upregulates TNF-α expression through MAPK signaling.

a Silencing of endogenous ANO1 promotes phosphorylation of ASK1, p38, JNK, and JUN. Immunoblots of lysates from prostate cancer PC-3 cells transfected with ANO1-siRNAs or NCsi for 72 h. b Bar graph showing quantitative analysis of protein expression from (a). Data were normalized to NC group cells (means ± SEM; n = 4). c MAPK inhibitors inhibit upregulation of TNF-α induced by ANO1 silencing. Immunoblots of lysates from PC-3 cells transfected by ANO1-siRNA3 or NCsi for 72 h and co-treated with 0.3 μM SCH772984 (ERK inhibitor), BIRB 796 (p38 inhibitor), or JNK-IN-8 (JNK inhibitor) for 24 h, respectively. d Bar graph showing quantitative analysis of protein expression from (c). Data are presented as the means ± SEM (n = 3). ^##p < 0.01 vs NC group. e, f BIRB 796 and JNK-IN-8 reverse the enhancement of TNF-α expression and apoptosis induced by ANO1 silencing. PC-3 cells were transfected by ANO1-siRNA3 or NCsi for 72 h and co-treated with 0.3 μM SCH772984, BIRB 796, or JNK-IN-8 for 24 h, respectively. e Apoptosis was assessed by Cell Death Detection ELISA^PLUS Kit. f Levels of TNF-α in culture supernatants were measured by ELISA. Data are presented as the means ± SEM (n = 5). ^##p < 0.01 vs NC group. *p < 0.05, **p < 0.01 vs siRNA3 group