Research Paper Volume 9, Issue 12 pp 2647—2665

Clock mediates liver senescence by controlling ER stress

Figure 6. Loss of PDIA3 promotes oxidative stress and apoptosis via the PERK pathway. (A) ROS activities were detected in the control siRNA (siCtrl)-treated and Pdia3 siRNA (siPdia3)-treated AML12 cells. After transfection for 24 hours, cells were exposed to 2 μg/mL TM for 6 hours; n=4 for all groups. (B) Relative Caspase 3 activity were detected in TM-treated AML12 cells. The cell treatment method was the same as in A. Caspase activity was measured in the cell lysate after addition of TM. The left panel shows the activity of the caspases after Pdia3 inhibition at a specific time point. The panel on the right shows the corresponding line graph. (n=3). **, P<0.01; *, P<0.05. (C) SOD, MDA, GSH and CAT activities were analyzed in AML12 cell homogenate treated as in (A) at the indicated times (0, 20, 40, and 60 minutes). CAT, GSH and SOD represent the antioxidation level; MDA represents the oxidation level. (n=4) **, P<0.01; *, P<0.05. (D) Relative expression assessed by qPCR of antioxidation genes (Cat, Sod, Sod2, Prdx1, Prdx2, gpx1, gpx2, gsr, and gstk1). Light gray represents the control group (siCtrl); dark gray represents Pdia3-inhibited group (siPdia3). Data were normalized to Gapdh expression. (n=4) **, P < 0.01 and *, P < 0.05 versus control. (E) AML12 cells were treated with the GSK inhibitor during the TM stress (2 μg/mL). Cell lysates were analyzed by the method used in (C) (n=4). **, P<0.01; *, P<0.05. (F) Relative activity of Caspase 3 was analyzed with the PERK inhibitor (GSK) and TM stress (2 μg/mL). (n=4) **, P < 0.01 and *, P < 0.05 versus control.