Fig 2: TNF-α expression is inversely correlated with ANO1 expression in prostate cancer cells.a–c Silencing of endogenous ANO1 increases TNF-α expression in PC-3 cells. a Representative immunoblots of ANO1 suppression by siRNAs and TNF-α upregulation. b Quantitative analysis of ANO1 and TNF-α protein expression from (a) (means ± SEM, n = 3). c Relative mRNA expressions of ANO1 and TNF-α was examined using qPCR and data are presented after being normalized to β-actin (means ± SEM; n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001 vs NC. d–f Normal prostate epithelium RWPE-1 cells stably transfected with ANO1 show a decrease of TNF-α protein and mRNA expression. Data are expressed as the means ± SEM (e n = 3; F: n = 4). * p < 0.05; ** p < 0.01 vs vector. g Immunostaining images in PC-3 cells treated with ANO1-siRNAs or NCsi for 72 h (magnification × 20). h Bar graph showing quantitative analysis of fluorescence intensity of TNF-α from (g). Data are presented as the means ± SEM; n = 4; ***p < 0.001 vs NC. i Silencing of endogenous ANO1 increases TNF-α production in PC-3 cells. Levels of TNF-α in culture supernatants were measured by ELISA. Parallel plates with the same treatment were used for cell counting. Data are expressed as the means ± SEM (n = 4). *p < 0.05; **p < 0.01 vs NC. j Lenalidomide reverses apoptosis induced by silencing ANO1. PC-3 cells were transfected with ANO1-siRNAs for 72 h and co-treated with 20 nM of lenalidomide for 24 h. Data are expressed as the means ± SEM (n = 4). * p < 0.05, **p < 0.01 for statistical significance for comparisons between lenalidomide versus vehicle groups. k Human TNF-α antibody reduces apoptosis induced by silencing ANO1. PC-3 cells were transfected with ANO1-siRNAs for 72 h and co-treated with 0.2 μg/ml of anti-TNF or normal IgG for 48 h. Data are expressed as the means ± SEM (n = 4). ** p < 0.01 for statistical significance for comparisons between human TNF-α antibody versus normal goat IgG. l–n ANO1 inhibitors, CaCCinh-A01 (l), T16Ainh-A01 (m), or Ani9 (n) increased TNF-α production in PC-3 cells. Levels of TNF-α in culture supernatants were measured by ELISA. Parallel plates with the same treatment were used for cell counting. Data are normalized and expressed as the means ± SEM (n = 4). *p < 0.05; **p < 0.01 vs control (0.1% DMSO)