Fig. 4

PERK pathway contributes to S4-induced immunogenic cell death in glioma cells. A LN229 cells treated with S4 (60 μM) or DMSO for 12 h were collected for transcriptome sequencing. Differentially expressed genes were analyzed by gene ontology. 18 differential genes related to ER stress were also listed. B Gene set enrichment analysis of S4-treated LN229 cells and cells treated with vehicle. C Real-time quantitative PCR analysis for DDIT3, DNAJB9, and SEL1L mRNA levels of S4-treated LN229 or U87MG cells and control cells. D Immunoblot (IB) analysis was performed to detect the action of the PERK pathway related proteins P-perk, ATF4, P-elf2α, and the IRE1α-XBP1 pathway related proteins pIRE1α, IRE1α, XBP1(s). E LN229 and U87MG cells were treated either IRE1α -xbp1 pathway inhibitor 4μ8C (50 μM), or PERK pathway inhibitors GSK2606414 (1 μM) and ISRIB (100 nM) with S4 (60 μM) for 24 h and the expression of ER stress related protein, HMGB1 and HSP70/90 was measured by IB analysis. F LN229 and U87MG cells were treated with S4 (60 μM) and GSK2606414 or ISRIB respectively, the CRT exposure was detected by confocal microscopy (scale bar = 25 μm). Arrowheads indicate positive area. G With or without S4 treatment, LN229 and U87MG cells were pretreated with GSK2606414 or ISRIB, and the media was collected to culture THP-1 cells. Release of IL-8 from THP-1 cells co-cultured with conditioned media was measured by ELISA. The release rate of control group was 100% for quantitative statistics. H LN229 and U87MG cells were treated with S4 (60 μM) and GSK2606414 or ISRIB respectively, expression of LC3II and cleaved-PARP was examined by IB analysis. The above experiments were performed three times (*P < 0.05, **P < 0.01, ***P < 0.001)