Fig. 4

Overexpressing TMEM120 accelerated breast cancer stemness by activating TAZ-mTOR signaling axis. (A) KEGG analysis was conducted to detect the signaling pathway significantly correlated with deletion of TMEM120B in MDA-453 cells. (B) Phosphorylation antibodies array kit was used to explore the key signaling pathway involved in TMEM120B overexpression in SK-BR-3 cells. (C) Immunoblotting of Myc-tag, TMEM120B, AKT, p-AKT, mTOR, p-mTOR, YAP, TAZ, and GAPDH after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. (D) qPCR assay was used to investigate the alteration of the target genes of YAP/TAZ within ectopic or deleted TMEM120B in SK-BR-3 or MDA-231 cells. (E) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without mTOR signaling pathway inhibitor rapamycin in SK-BR-3 cells. (F) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120 with or without knocking down TAZ by siRNA in SK-BR-3 cells. Subcellular localization of TAZ was evaluated by immunofluorescence assay (G) or western blotting assay (H) within ectopic TMEM120B in SK-BR-3 cells. Scale bar = 20 μm. (I) After being treated with CHX at indicated time points, the expression of TAZ was evaluated by western blotting after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. Quantification data are expressed as mean ± SD of three independent experiments (t-test, two-sided, ***, P < 0.001)