Research Paper Volume 9, Issue 12 pp 2647—2665

Clock mediates liver senescence by controlling ER stress

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.