Fig. 4: RGS6 blocked activation of downstream effectors of TGF-β-induced pro-EMT signaling.

A After being serum starved for 24 h, RGS6-Cas9 NSCLC cells and control cells were treated with or without TGF-β (5 ng/ml) for 24 h. Western blotting was performed to detect expression of EMT-related markers and effectors E-cadherin, N-cadherin and Snail. Noted that there is no E-cadherin expression in H1299 cells. B RGS6-Cas9 NSCLC cells and control cells were treated with or without TGF-β1 (5 ng/ml) for 2 h, and subjected to qRT-PCR for detection of Snail mRNA levels (*p < 0.05, **p < 0.01). C After being serum starved for 24 h, RGS6-HA stable NSCLC cells and control cells were treated with or without TGF-β (5 ng/ml) for 24 h. Western blotting was performed to detect expression of E-cadherin, N-cadherin and Snail. D RGS6-HA stable NSCLC cells and control cells were treated with or without TGF-β1 (5 ng/ml) for 2 h, and subjected to qRT-PCR for detection of Snail mRNA levels (**p < 0.01). E RGS6-HA A549 cells and control cells were treated with TGF-β (5 ng/ml) for indicated time periods, and subjected to qRT-PCR for detection of PAI-1 mRNA levels (**p < 0.01, ***p < 0.001). F The PAI-1 promoter constructs were transiently transfected into RGS6-HA and control A549 cells, and the relative luciferase activities were determined using the Dual-Luciferase reporter assay system after being treated with TGF-β (5 ng/ml) for 24 h (**p < 0.01). G RGS6 level is negatively correlated with PAI-1 expression in LUAD patients from TCGA dataset. X and Y axes represent levels of RGS6 and PAI-1 mRNA, respectively. H RGS6 level is negatively correlated with SNAI1 gene expression in LUAD patients from TCGA dataset. X and Y axes represent levels of RGS6 and SNAI1 mRNA, respectively.