Figure 6. TR4 contributes to the chemosensitivity in HCC cells through up-regulation of ATF3 expression.

(A) Western blot analysis results show lower expression of ATF3 and cleaved PARP in TR4 knockdown Huh7 cells. Expression of TR4, ATF3, and cleaved PARP in Huh7-shTR4/Huh7-scr cells were shown. (B) Western blot analysis results show higher expression of ATF3 and cleaved PARP in TR4 overexpression HCC cells. Expression of TR4, ATF3, cleaved PARP in LM3-shTR4/LM3-scr cells were shown. (C, D) Effect of ATF3 interruption in neutralizing enhanced chemosensitity induced by TR4 overexpression in LM3 cells and SNU387 cells respectively. Western blot analysis of TR4 and ATF3 protein levels in LM3 cells, infected with either shATF3 or vector. Cells were infected by siATF3 and then treated with indicated concentration of cisplatin for 48 h. Cell viability was analyzed by MTS assays. (E, F) Q-PCR analysis results show lower expression of ATF3 in TR4 knocked down Huh7 cells and higher expression of ATF3 in TR4 overexpressed LM3 cells. (G) TR4 directly binds to the promoter of ATF3 through ChIP assay. Huh7 cells were cross-linked with formaldehyde (final 1%) for 15 min at room temperature and then subjected to ChIP assay using an anti-TR4 antibody or IgG (negative control) and the indicated primers. Reaction products were resolved by electrophoresis. The values presented are the means ± SD for each group (H) Huh7 cells were cultured and transiently transfected with ATF3-luciferase reporter without or with knocking down TR4 expression. (I) LM3 cells were cultured and transiently transfected with ATF3-luciferase reporter without or with overexpressing TR4 expression. (*P < 0.05, **P < 0.01).