PER2 Regulates Reactive Oxygen Species Production in the Circadian Susceptibility to Ischemia/Reperfusion Injury in the Heart

Figure 4

PER2 deficiency in the tolerance of H9c2 cells to H₂O₂ treatment after serum shock. (a) Per2 mRNA expression during 24 h after serum shock ( per time point, vs. 0 h after serum shock, one-way ANOVA with Bonferroni correction). (b) Per2 mRNA expression levels in H9c2 cells after knockdown of Per2 using siRNA (, vs. siRNA-Sc-0 hr, ^# vs. siRNA-Sc-12 hr). (c–g) H9c2 cells were transfected with siRNA-Sc or siRNA-Per2 24 h before serum shock. Transfected cells were treated with H₂O₂ for 2 h at the ending point or 12 h after serum shock. (c) ATP change in transfected H9c2 cells treated with H₂O₂ at 0 h and 12 h after serum shock. (d) Cell death determined by TUNEL staining. Representative image (left) and quantification (right). (e) ROS in cells. Flow cytometry images (upper), quantitative analysis of ROS (lower panel, left figure). (f) Flow cytometry of JC-1 in cells. Representative images (f), quantitative analysis of JC-1 ((e), lower panel, right figure). (g) Per2 and potential target levels in H9c2 cells were determined by RT-PCR at 0 h and 12 h. Quantification of Per2, Cpt1a, and Pdhb was normalized to GAPDH (, , , and vs. cells transfected with siRNA-Sc at 0 h after serum shock; ^#, ^##, and ^#### vs. cells transfected with siRNA-Sc at 12 h after serum shock; ^& and ^&&&& vs. cells transfected with siRNA-Sc treated with H₂O₂ at 0 h after serum shock; ^$$$$ vs. cells transfected with siRNA-Sc with H₂O₂ treatment at 0 h after serum shock; ^{^^^^} vs. cells transfected with siRNA-Sc with H₂O₂ treatment at 12 h after serum shock; ^!!!! vs. cells transfected with siRNA-Per2 with H₂O₂ treatment at 0 h after serum shock). Alpha was set as 0.05. Data are presented as the . The Holm-Sidak method was used to correct for multiple -tests.

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