Bmal1 Regulates the Redox Rhythm of HSPB1, and Homooxidized HSPB1 Attenuates the Oxidative Stress Injury of Cardiomyocytes

Figure 3

Homooxidized HSPB1 was necessary for HSPB1 to resist oxidative stress damage. (a, b) The pEnCMV-HSPB1C141S^mut or vector was transfected into the H9c2 cells with HSPB1 stable knockout for 48 h; flow cytometric analysis of cell apoptosis was conducted. (a) Representative plots of flow cytometric analysis are presented. (b) Data are presented as the means ± standard deviation (). (c–f ) The pEnCMV-HSPB1C141S^mut or vector was transfected into the H9c2 cells with HSPB1 stable knockout for 48 h; (c, d) cells were then treated with 800 mM H₂O₂ for 24 h. Western blot analysis was used to determine the levels of cleaved caspase-3; (e) H9c2 cells transfected with pCMV‐myc‐HSPB1 or vector for 48 hr and then incubated with CM‐H2DCFDA dye at a concentration of 10 μM for 30 min at 37°C. Cells were then treated with 0.4 mM H₂O₂ for the indicated time periods (15, 30, 60, and 120 min). Fluorescence was quantified using flow cytometry with excitation at 485 nm and emission at 530 nm (). (f–h) Intracellular total GSH (f) and GSSG (g) levels were determined by glutathione reductase recycling assays. The reduced GSH/GSSG ratio (h) was calculated using reduced and oxidized GSH concentrations. Values represent the means ± standard deviations (; ; ).

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