Fig. 3

ER stress induced XBP1 upregulates PDIA4 expression transcriptionally. A-B RT-qPCR quantified the relative mRNA expressions of PDIA4, XBP1 and ATF6 in TM-induced ER stressed U87 (A) and LN229 (B) cells at 0, 6, 12 and 24 h. *P < 0.05; **P < 0.01; ***P < 0.001. C Western blot assay showed the ATF6, XBP1-s, XBP1-u and PDIA4 protein expressions were all upregulated under TM-induced ER stress in GBM cells. D-E Blocking XBP1 in ER stressed GBM cells by siRNAs significantly downregulates PDIA4 mRNA expressions. ***P < 0.001. F Upregulation of PDIA4 protein under ER stress was intercepted by blocking XBP1 expressions in GBM cells. G Design of ChIP-PCR primers of XBP1 binding regions. H-I The ChIP-PCR results showed Region 2 sequence (-1215 ~ -1202) on PDIA4 promoter were captured by XBP1 protein immunoprecipitation in U87 (H) and LN229 (I) cells. ns P > 0.05; ***P < 0.001. J The DNA gel electrophoresis shows the abundance of PCR produced DNA sequences of ChIP assay. K The luciferase activities of the vector, full length, region 2 and mutant PDIA4 promoter sequence transfected LN229 cells with or without XBP1 knock-down under 10 μg/mL TM induced ER stress. ns P > 0.05; ***P < 0.001. L The luciferase activities of the vector, full length, region 2 and mutant PDIA4 promoter sequence transfected U87 cells with or without XBP1 knock-down under 10 μg/mL TM induced ER stress. ns P > 0.05; ***P < 0.001