HSP72 Protects Cells from ER Stress-induced Apoptosis via Enhancement of IRE1α-XBP1 Signaling through a Physical Interaction
Figure 7
Regulation of IRE1α-XBP1 by Hsp72 contributes to thermotolerance against ER stress and increased secretion of neurotrophins.
(A) PC12 cells were transduced with lentivirus expressing control non-targeting shRNA or 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. (B) The control (PGIPZ) or XBP1 shRNA expressing (XBP1 shRNA) PC12 cells were heat shocked for 1 h at 42°C and left to recover for 6 h. Western blots on whole cell lysates were carried out to check the expression of Hsp72 after heat shock with β-actin as loading control. (C) Normal (PGIPZ C, XBP1shRNA C) and thermotolerant (PGIPZ HS, XBP shRNA HS) control and XBP1 shRNA expressing PC12 cells were either 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 MTT assay. Average and error bars represent mean ± SD from three independent experiments performed in triplicate. (D–E) The control (Neo) and Hsp72 expressing (Hsp72) PC12 cells were either untreated (Un) or treated with (0.1 µM) Tg, (0.5 µg/ml) Tm, or (50 µM) 6-OHDA for 24 h. Culture supernatant was analyzed for NGF and BDNF according to the conditions as described in Materials and Methods. Average and error bars represent mean ± SD from three independent experiments performed in triplicate. * indicates a statistical significance between Neo and Hsp72 cells; p<0.05. ** indicates a statistical significance between Neo and Hsp72 cells; p<0.005.