Fig. 5

TMEM120B promoted breast cancer cell stemness by binding with MYH9 via their coil-coil domains. (A) Mass spectrometry (MS) analysis was performed to identify candidates for interaction with TMEM120B after overexpressing TMEM120B in SK-BR-3 cells. Endogenous (B) and exogenous co-IP assay (C) were assessed to detect the interaction between MYH9 and TMEM120B in MDA-231 and SK-BR-3 cells. (D) GST pull-down assay was used to confirm the direct binding between MYH9 and TMEM120B in SK-BR-3 cells. (E) Immunofluorescence assay was used to show the co-localization of TMEM120B and MYH9 in SK-BR-3 cells, subcellular location coefficient of TMEM120B–MYH9 interaction was quantified by Fiji software (scale bar = 50 μm) (F) Divergent TMEM120B and MYH9 splicing mutant plasmids were designed to examine the domain responsible for the interaction between TMEM120B and MYH9.(I) GST pull-down assay was performed to confirm the direct interaction between MYH9 and TMEM120B after overexpressing TMEM120B-WT or TMEM120B-∆CCD plasmids in SK-BR-3 cells. Colony formation assay (J), Transwell assay (K), and sphere formation assay (L) were performed to detect the effects on the proliferation, invasion, and stemness of breast cancer cells after transfecting TMEM120B, TMEM120B-∆CCD, and control plasmid in SK-BR-3 cells. (M) Immunoblotting of Myc-tag, mTOR, p-mTOR, TAZ, and GAPDH after overexpressing TMEM120B or TMEM120B-∆CCD in SK-BR-3 cells. (N) Immunoblotting was used to evaluate the expression of cytosolic or nuclear TAZ after overexpressing TMEM120B or TMEM120B-∆CCD in SK-BR-3 cells. The effects on proliferation, metastasis, and stemness were verified in nude mice by subcutaneous tumorigenesis (O), tail vein injection (P), and diluted injection xenografts assays (Q) by overexpressing TMEM120B and TMEM120B-∆CCD in SK-BR-3 cells. Quantification data are expressed as mean ± SD of three independent experiments (t-test, two-sided, **, P < 0.01, ***, P < 0.001)