Effect of RCAN1.4 silencing on activated HSC-T6 cells. (A) Protein and (B) mRNA expression of RCAN1.4, COL1a1, and α-SMA. (C) Relative cell viability of HSC-T6 cells was measured by CCK-8 analysis. (D) Cell cycle was determined by flow cytometry. (E) The cell-cycle-related proteins C-Myc and Cyclin D1 levels were analyzed by western blotting analysis. (F) Effect of RCAN1.4 silencing on apoptosis of activated HSC-T6 cells was detected by flow cytometry. (G) The expression levels of apoptosis-associated proteins (Bax, Bcl2 and cleaved-caspase3 protein) were detected by western blotting assay. Representative images of three independent experiments are shown. ***p<0.001, #p<0.05, ###p<0.001, as indicated. For all panels, data represent the mean ± s.e.m. for 3-4 independent experiments in vitro.