Cell fate determined by the activation balance between PKR and SPHK1

摘要

a The levels of phosphorylated PKR and SPHK1 were evaluated following treatment with 2 μM or 400 nM DON for the indicated time periods in HepG2 and HEK293T cells. The cells were then harvested and subjected to western blotting analysis. The dosages of DON administrated in both cells are chosen according to IC50 measurements by CCK-8 assay as shown in Supplementary Fig. 1. b The levels of phosphorylated PKR and SPHK1 were evaluated following irradiation with UVC for 10 min, treatment with 10 ng/mL TNF-α, or treatment with 1 μg/mL LPS for 3 h in HepG2 and HEK293T cells. The cells were then harvested and subjected to western blotting analysis. c The expression of ER stress-related proteins were evaluated during PKR overexpression and PolyI:C administration. HEK293T cells were transfected with pCDNA3.1–PKR or pCDNA3.1 for 24 h or treated with 10 μg/mL PolyI:C for 3 h. The optimization of PolyI:C concentrations as the positive control is shown in Supplementary Fig. 5. The cells were then harvested and subjected to western blotting analysis. d, e ER stress-related protein levels during DON or TNF-α exposure were evaluated in PKR knockout cells. HEK293T control and PKR knockout cells at 70% confluence were incubated with 400 nM DON or 10 ng/mL TNF-α for 3 h. The cells were then harvested and subjected to western blotting analysis. f Cytotoxicity of DON was evaluated in PKR knockout cells. HEK293T control and PKR knockout cells at 70% confluence were treated with various concentrations of DON for 24 h. Cell viability was then determined with CCK-8 (n = 6). g Cell apoptosis resulting from TNF-α treatment in PKR knockout cells. HEK293T control and PKR knockout cells were evaluated with 10 ng/mL TNF-α by targeting cleaved caspase 9. The cells were then harvested and subjected to western blotting analysis. h The expression levels of IKKα and phosphorylated MAPKs during SPHK1 overexpression. HepG2 cells were transfected with pCDNA3.1–SPHK1 or pCDNA3.1 for 24 h. The cells were then harvested and subjected to western blotting analysis. i, j The expression levels of DON- and UV-induced IKKα and phosphorylated MAPKs were evaluated in shSPHK1 cells. HepG2 shLacZ and shSPHK1 cells at 70% confluence were incubated with 2 μM DON for 3 h or exposed to UVC irradiation for 10 min. The cells were then harvested and subjected to western blotting analysis. k Cytotoxicity of DON in shSPHK1 cells. HepG2 shLacZ and shSPHK1 cells at 70% confluence were treated with various concentrations of DON for 24 h. Cell viability was then determined with CCK-8 (n = 6). l Cell apoptosis resulting from TNF-α treatment in shSPHK1 cells. HepG2 shLacZ and shSPHK1 cells were evaluated with 10 ng/mL TNF-α by flow cytometry. The data were analyzed using FlowJo software. The results are the means ± SEMs of at least three independent experiments. Statistical significance was defined as *p < 0.05, **p < 0.01, or ***p < 0.001.