Fig. 3: ASK1 functions as a key mediator of TGF-β downregulation-induced cell death triggered by non-canonical signaling Akt inactivation

a ASK1 mediates TGF-β-induced cell death via p38/JNK activation. A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 (100 MOI) and subsequently transfected with siASK1. siASK1 transfection was based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, cell viability was tested by an MTS viability assay. Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (*p < 0.05; **p < 0.01). b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with siASK1. After 48 h, morphological changes were observed using microscopy. c A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with siASK1. After 48 h, the expression levels of p-p38, p-Akt, p-HSP27, HSP27, p-ERK, p-src, p-p65, p-JNK, p-stat3, and GAPDH were detected by western blot analysis. d A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with ASK1 kinase mutant ASK1 (K709M). After 48 h, cell viability was tested with an MTS viability assay. Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (*p < 0.05; **p < 0.01). e A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with ASK1 kinase mutant ASK1 (K709M). After 48 h, morphological changes were observed using microscopy. f A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with ASK1 kinase mutant ASK1 (K709M). After 48 h, the expression levels of p-p38, p-Akt, p-HSP27, HSP27, p-ERK, p-src, p-p65, p-JNK, p-stat3, and GAPDH were detected by western blot analysis. g A375 cells were infected with adenovirus expressing shTGF-β1 and subsequently transfected with siSmad4. After 48 h of incubation with DCF-DA (20 μM, 1 h), ROS were detected using a fluorescent reader and microscopy. h After 48 h, the expression of various ER stress markers in addition to survival-related molecules was detected by western blot analysis; A375 cells were infected with adenovirus expressing shTGF-β1 and subsequently transfected with pCMV6-myr-Akt. i After 48 h of incubation with DCF-DA (20 μM, 1 h), ROS were detected using a fluorescent reader and microscopy. j After 48 h, the expression levels of various ER stress markers and phospho-Akt and Akt were detected by western blot analysis. k After 48 h, the expression levels of PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, JNK, and GAPDH were detected by western blot analysis. l After 48 h, cell viability was tested via an MTS viability assay. Error bars represent the standard error from three independent experiments. Decreased p value to 0.041 from <0.001 after Akt activation followed by TGF-β1 downregulation means a significant decrease in cell death