Research Paper Volume 9, Issue 12 pp 2647—2665

Figure 5. The lack of Pdia3 leads to activation of PERK but not IRE or ATF6. (A-B) Control siRNA (siCtrl)- and Pdia3 siRNA (siPdia3)-treated AML12 cells were exposed to 2 μg/mL TM for the indicated times, and GSK2606414 (PERK inhibitor) was introduced with TM treatment at the indicated concentration for 7 hours. Cell lysates were then immunoblotted to detect (p)PERK, (p)eIF2α, eIF2α and PDIA3. In addition, the mean ± S.E.M. of the phosphorylated /total eIF2a ratio (normalized to eIF2a) is shown in (B) (n=4). **, P<0.01; *, P<0.05. (C) Immunoblotting of cells from siCtrl- and siPdia3-treated AML12 cells to show the level of PDIA3 depletion. Note that no change was found in levels of PDI or PDIA6 (n=4). (D-E) Immunoblots (D) and relative mRNA expression (E) of IRE1α in siCtrl- and siPdia3-treated AML12 cells. No significant difference was found. (n=4); **, P<0.01; *, P<0.05. (F) Endogenous immunoprecipitation of PDIA3 pulled down endogenous IRE1α from HEK293T cell lysates; 1% of lysate was used as the input control (n=3 experiments). (G) siCtrl- or siPdia3-treated AML12 cells were incubated with 2 μg/mL TM for the indicated times, and un-spliced (u) and spliced (s) XBP1 mRNA was amplified by qPCR. Gapdh served as the control. Means ± S.E.M are plotted. **, P < 0.01 and *, P < 0.05 versus control. n=4 mice per group. (H-I) Immunoblots (H) and relative mRNA expression (I) of ATF6 in siCtrl- and siPdia3-treated AML12 cells. (n=4) **, P < 0.01 and *, P < 0.05 versus control.