Figure 4

Characterization of U87Δ899 IRE1 RNase dominant-negative cells. (a) The U87Δ899 RNase construct was designed to express an IRE1α protein truncated at its cytoplasmic C-terminal end in the RNase domain. (b) Inhibition of XBP1 splicing in three different U87Δ899 RNase clones (R2, R3 and R7, upper panel) and in U87dn cells (lower panel). Cells were stimulated for 2 h with 10 μg/ml tunicamycin/DMSO (Tun) or with DMSO only. Amplification of XBP1 transcripts was carried out after reverse transcription using primers flanking the XBP1 mRNA splicing sites. PCR products were analyzed by electrophoresis on 2% agarose gels. XBP1s and XBP1u represent spliced and unspliced mRNA, respectively. (c) MIST transcripts were measured by qPCR in U87wt, U87Ctrl, U87dn and U87Δ899 cells subjected or not to tunicamycin treatment for 16 h. The reference value (1.00) corresponds to the value obtained with U87wt cells in the absence of tunicamycin. Results were normalized using HPRT1 mRNA as standard. qPCR was performed in triplicate on three independent RNA preparations. Data are presented as mean ± SD. (d) IRE1 kinase autophosphorylation in U87Δ899 cells. Immunoblotting analysis of total IRE1α (IRE1) and of phospho-Ser724-IRE1 (p-IRE1) proteins after a 2h-incubation with or without tunicamycin.