Fig. 5

ROS upregulates FOXC1 expression through ERK1/2-p-ELK1 pathway. a After the cells were treated with BSO (30 μM, 24 h) or NAC (0.2 mM, 24 h), the ROS levels were detected by immunofluorescence. b After the cells were treated with BSO (30 μM, 24 h) or NAC (0.2 mM, 24 h), the migration and invasion abilities of the indicated HCC cells were detected by Transwell assays. c CCK8 assays indicated that NAC (upper panel) abolished FOXC1-mediated cell proliferation and BSO (lower panel) rescued cell proliferation inhibition by FOXC1 knockdown. d Immunofluorescence detection of the ROS levels when PLC/PRF/5 and Huh7 cells were preprocessed with BSO (30 μM, 24 h) or NAC (0.2 mM, 24 h). e After PLC/PRF/5 and Huh7 cells were treated with BSO (30 μM, 24 h) or NAC (0.2 mM, 24 h), western blot was used to detect the protein level of FOXC1. f Huh7 cells followed by BSO disposing (30 μM, 24 h) were transfected with FOXC1 promoter construct, and then to examine the relative luciferase activity. g Continuous truncated and mutated FOXC1 promoter constructs were transfected into cells stimulated by BSO to determine the relative activity of luciferase. h ROS induces FOXC1 expression by activating ELK1. Huh7 cells followed by BSO disposing and infected with the lentivirus LV-shELK1 were transinfected with the FOXC1 promoter construct, and then use luciferase reporter assays to detect the relative luciferase activity. i Huh7 cells were treated with BSO and then transfected with lentivirus LV-shELK1 to inhibit the expression of ELK1. After treated with BSO for twenty-four hours, western blotting analysis detected the protein levels of FOXC1 expression. j The protein levels of total and phosphorylated Akt, ERK1/2, JNK, p38 and p65 and FOXC1 expression upon BSO stimulation and indicated inhibitor treatment were analyzed by western blot. (K) ChIP assays demonstrated that ROS promoted the direct binding of ELK1 to the FOXC1 promoter through the ERK1/2 pathway. Data are represented as mean ± S.D. of three experiments. *P < 0.05