Research Paper Volume 9, Issue 12 pp 2647—2665

Clock mediates liver senescence by controlling ER stress

Figure 7. Pdia3 can reverse the aging process of the liver in ClockΔ19 mice. (A) AML12 cells were transfected with the pcDNA3.1-Pdia3 plasmid (Trans gene, TG) for 24 hours and then stimulated with TM stress (2 μg/mL) for 2 hours. SOD, MDA, GSH and CAT activity were analyzed in the cell homogenate. (n=4) **, P<0.01; *, P<0.05. (B) AML12 cells were treated as described in (A), and the activity of Caspase 3 was measured to reflect apoptotic activity. (n=4) **, P < 0.01 and *, P < 0.05 versus control. (C) SAHF stained via DAPI were visualized by fluorescence microscopy. The primary hepatocytes were extracted, and the number of SAHF was observed after transfection with the pcDNA3.1-Pdia3 plasmid for 24 hours. The SAHF were greatly rescued in ClockΔ19+Pdia3(TG) cells (n=3). (D) The primary liver cells were extracted and transfected with the pcDNA3.1-Pdia3 plasmid for 48 hours. Immunoblotting methods were used to detect the expression of UPR proteins (p)PERK and (p)eIF2α, PDIA3, senescent protein P53, and DNA damage protein γ-H2AX. The statistical results are displayed (right). The results are expressed as the mean ± S.E.M (n=4). **, P < 0.01 and *, P < 0.05. (E) Primary liver cells were extracted and treated as described in (D). Then, the relative activity of Caspase 3 was detected to reflect the level of apoptosis. Note that the apoptotic activity was significantly reduced after transfection with Pdia3. (n=4) **, P<0.01; *, P<0.05. (F) Immunoblots showing the expression levels of γH2AX, PARP, P53, (p)PERK, (p)eIF2a and PDIA3 in the WT, ClockΔ19 and ClockΔ19 heterozygote groups (36 week old). n=3 for all groups. (G-H) Histological analysis of mice as described in (F). HE and SA-β-gal staining of the liver tissue. (n=3 for all groups). Note that the ClockΔ19 HET mice show a decreased inflammation foci and improved aging phenotype.