The significance of IRE1α-XBP1 pathway in MM cells. (A) Expression of IRE1α in MM cell lines was detected by Western blotting. Actin served as a loading control. (B) XBP1 mRNA was detected by RT-PCR. XBP1u was observed as a 152-bp band, and XBP1s was observed as a 126-bp band. β-actin served as a loading control. (C-E) RPMI 8226 cells were transduced with 2 independent shRNA lentivirus vectors (XBP1sh#1 and XBP1sh#2) to knockdown XBP1 gene expression. A vector-targeting luciferase was used as a control. (C) Three days after transduction, total RNA was extracted, and XBP1 mRNA was evaluated by real-time quantitative PCR in the graph. Data represent mean ± SD fold changes relative to β-actin mRNA in triplicate samples. Expression of XBP1u and XBP1s was examined by RT-PCR in the right panel. (D) Two days after transduction, cells were incubated for the indicated times. Growth of the cells was assessed by MTT assay of quadruplicate cultures, expressed as a percentage of 4-hour samples. Data represent mean ± SD. (E) Two days after transduction, cells were treated with bor-tezomib (4nM) or 17-AAG (250nM) for 48 hours. Cell proliferation was assessed by [³H]-thymidine uptake of quadruplicate cultures, expressed as a percentage of untreated control vector cells. Data represent mean ± SD.