Fig. 3

IRE1 I642G confers endoribonuclease activity and induces proliferation. a (top) Control (Puro) and PC-3 cells stably expressing the I642G IRE1 construct were subjected to Western blot analysis to detect IRE1 expression. (bottom) Control (Puro) and PC-3 cells stably expressing the I642G IRE1 construct were treated with DMSO, 1NM-PP1 (5 µM), or thapsigargin (Tg, 300 nM) for 24 h and Xbp-1 splicing examined. b PC-3 cells (Puro and IRE1-I642G) were treated with Velcade and thapsigargin in the presence or absence of 1NM-PP1 (5 µM) for 24 h and cell viability quantified with an MTT assay. Values depict the average ± the standard deviation. c Expression of CHOP and Grp78 mRNA in PC-3 cells (Puro and IRE1-I642G) treated with DMSO or 1NM-PP1 (5 µM) for 24 h as determined by quantitative real-time PCR. Values were standardized and compared to Puro cells treated with DMSO only. Error bars reflect the standard deviation. d (top) Control (Puro) and DU145 cells stably expressing the I642G IRE1 construct were subjected to Western blot analysis to detect IRE1 expression. (bottom) Puro and DU145 cells stably expressing I642G IRE1 were treated with DMSO, 1NM-PP1 (5 µM), or thapsigargin (Tg, 300 nM) for 24 h and Xbp-1 splicing examined. (e) DU145 cells (Puro and I642G IRE1) were treated and the results depicted as described in b. f BrdU labeling was carried out on PC-3 (left) and DU145 (right) cells expressing the I642G IRE1 construct or vector only (Puro). 1NM-PP1 (5 µM) was added to the PC-3 cells for 24 h. BrdU labeling in the DU145 cells as a function of serum concentration was conducted in the absence of 1NM-PP1. The average of three replicates ± the standard deviation is shown. gXbp-1 splicing status in PC-3 cells stably expressing the IRE1 K599A, or I642G, or empty pBabe Puro construct. PCR reactions were analyzed by polyacrylamide gel electrophoresis. Note, a reduction in sXbp-1 levels in the K599A cells and an induction of the spliced Xbp-1 message in the I642 cells compared to the Puro control