Figure 4. Increased production of spliced XBP1 contributes to cytoprotective function of Hsp72 against ER stress-induced apoptosis.

(A) Schematic presentation of wild-type and mutant IRE1α plasmids. (B) PC12 cells were transfected with indicated IRE1α plasmids. 24 h post-transfection, cells were either untreated (Un) or treated with (0.25 µM) Tg for 6 h. RT-PCR analysis of total RNA was performed to simultaneously detect both spliced and unspliced XBP1 mRNA and GAPDH. The image is presented inverted for greater clarity. (C) pCMV.SPORT-βGAL was co-transfected with either pcDNA3.1 or IRE1α ΔRNase expression plasmid in control (Neo) or Hsp72 expressing (Hsp72) PC12 cells. 24 h post-transfection, cells were either left untreated (Un) or treated with (0.25 µM) Tg for 48 h, (2 µg/ml) Tm for 48 h, (150 nM) staurosporine (STS) for 16 h, or (25 µg/ml) etoposide (ETOP) for 24 h. The reduction in cell viability was determined by measuring the reduction in β-galactosidase activity after the drug treatments. Average and error bars represent mean ± SD from three independent experiments performed in triplicate. ** indicates a statistical significance between Hsp72 and Hsp72 plus IRE ΔRNase cells; p<0.005. (D) Hsp72 expressing PC12 cells were transduced with lentivirus expressing indicated XBP1 targeting shRNA. RT-PCR analysis of total RNA was performed to simultaneously detect unspliced XBP1 mRNA and GAPDH. The image is presented inverted for greater clarity. (E) The control (Neo), Hsp72 expressing (Hsp72), and Hsp72 cells expressing indicated shRNAs were either untreated (Un) or treated with (0.25 µM) Tg, (2 µg/ml) Tm, (150 nM) staurosporine (STS), or (25 µg/ml) etoposide (ETOP). The reduction in cell viability was determined by MTT assay. Average and error bars represent mean ± SD from three independent experiments performed in triplicate. ** indicates a statistical significance between Hsp72 and Hsp72 plus XBP1 shRNA cells; p<0.005.