Fig. 6: Involvement of reciprocal ER stress and SAPK activation during TGF-β downregulation-induced cell death.

a A375 cells were infected with adenovirus expressing shTGF-β1 or -β2. After 48 h, various ER stress-related proteins were detected by western blot analysis. b A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h. Then cell viability was tested using an MTS viability assay (left). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (**p < 0.01). Additionally, the expression levels of PARP and GAPDH were detected by western blot analysis (right). c A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h. Then ER stress-related proteins that responded to TGF-β downregulation (Fig. 5a) were detected by western blot analysis. d A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h and then incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy. e A375 cell lines were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h. Then lysates were subjected to immunoprecipitation using an anti-Trx or anti-GSTM1 antibody to identify changes in ASK1. f A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h. Then the expression levels of PARP, ASK1, p-ASK1, p-p38, p38, p-JNK, JNK, Trx, GSTM1, and GAPDH were detected by western blot analysis