Fig. 7

TMEM120B–MYH9 interaction activated the TAZ-mTOR axis by accelerating FAK assembly. (A) GO analysis for TMEM120B interaction candidates from MS analysis after overexpressing TMEM120B in SK-BR-3 cells. (B) Venn analysis for the overlap between RNA-seq and MS analysis. (C) 3D collagen gel invasion assay was performed after overexpressing TMEM120B or TMEM120B-∆CCD and control in MDA-231 cells. (D) Immunoblotting assay was performed to evaluate the expression of Myc-tag, FAK, p-FAK (Tyr397), β1-integrin, active-β1-integrin, and LaminB1 after transfecting TMEM120B-myc, TMEM120B-∆CCD-myc, and control plasmids in SK-BR-3 cells. (E) Immunoblotting of Myc-tag, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120B with or without FAK signaling pathway inhibitor PF562271 in SK-BR-3 cells. Transwell assay (F), sphere formation assay (G), and colony formation assay (H) were performed to detect the effect on the invasion, stemness, and proliferation of breast cancer cells upon ectopic TMEM120B or ∆TMEM120B-CCD in SK-BR-3 cells. Representative immunofluorescence images of p-FAK (I) and β1-integrin (J) after treatment with nocodazole (NZ), followed by washout for 0, 30, and 60 min. Scale bar = 10 μm. (K) Immunoblotting of Myc-tag, Flag-tag, FAK, p-FAK(Tyr397), mTOR, p-mTOR, TAZ, ALDH1, and GAPDH after transfected with TMEM120B-myc, TMEM120B-∆CCD-myc, MYH9-flag, MYH9-delCCD-flag alone, or TMEM120B-myc + MYH9-flag, TMEM120B-∆CCD-myc + MYH9-flag, or TMEM120B-myc + MYH9-delCCD-flag in SK-BR-3 cells, respectively. Quantification data are expressed as mean ± SD of three independent experiments (t-test, two-sided, *P < 0.05, **P < 0.01, ***P < 0.001)