Cul4a as a New Interaction Protein of PARP1 Inhibits Oxidative Stress-Induced H9c2 Cell Apoptosis

Figures

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  Figure 1. H2O2 increased Cul4a and apoptosis-related protein and decreased the viability of H9c2 cardiomyocytes. (a) H9c2 cells were untreated or treated with 50 μM, 100 μM, 200 μM, and 400 μM H2O2 for 2 h. Cell viability was assessed by CCK-8 assay. (b) Flow cytometry Annexin-FITC/PI showed that the apoptosis rate of cell increased gradually induced by gradient concentration of H2O2. (c) Immunofluorescence staining of H9c2 with Cul4a antibody (green) and DAPI for nuclei (blue) after treatment with 200 μM of H2O2 for 2 h. (d) Western blot analysis of Cul4a, cleaved PARP1 and cleaved caspase3 expression after incubation with H2O2 (50 μM, 100 μM, 200 μM, and 400 μM) for 2 h; α-tubulin was used as a loading control. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05; scale bar, 50 μm. Data was expressed as means ± SD.

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  Figure 2. Cul4a overexpression inhibited H2O2-induced cardiomyocyte apoptosis. (a) H9c2 cells were untreated or treated with 50 μM, 100 μM, 200 μM, and 400 μM H2O2 for 2 h. CCK-8 assay results showed that overexpressed Cul4a decreased H2O2-induced cell cytotoxicity. (b) Flow cytometry Annexin-FITC/PI showed that overexpressed Cul4a reduced H2O2-induced cell apoptosis. (c) Overexpressed Cul4a significantly decreased H2O2-induced apoptotic marker proteins cleaved PARP1 and cleaved caspase-3 expression by western blot assay; α-tubulin was used as a loading control. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. Data was expressed as means ± SD. Cul4a: cullin 4a; PARP1: poly (ADP-ribose) polymerase-1.

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  Figure 3. Cul4a knockdown aggravated H2O2-induced H9c2 cell apoptosis. (a) Efficient Cul4a knockdown was established successfully when compared to control siRNA-transfected cells, and Cul4a siRNA target sequence 2 has the highest knockdown efficiency. (b) H9c2 cells were untreated or treated with 50 μM, 100 μM, 200 μM, and 400 μM H2O2 for 2 h. CCK-8 assay results showed that Cul4a knockdown increased H2O2-induced cell cytotoxicity. (c) Flow cytometry Annexin-FITC/PI showed that Cul4a knockdown aggravated H2O2-induced cell apoptosis. (d) Knockdown Cul4a significantly enhanced H2O2-induced apoptotic marker proteins cleaved PARP1 and cleaved caspase-3 expression by western blot assay; α-tubulin was used as a loading control. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. Data was expressed as means ± SD. Cul4a: cullin 4a; PARP1: poly (ADP-ribose) polymerase-1.

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  Figure 4. Cul4a influenced H2O2-induced ROS formation. (a) H9c2 cells were untreated or treated with 50 μM, 100 μM, and 200 μM H2O2 for 2 h; ROS production increased gradually by using fluorescence microscopy. (b) Cul4a overexpression inhibited H2O2-induced ROS production by using fluorescence microscopy. (c) Cul4a knockdown enhanced H2O2-induced ROS production by using fluorescence microscopy. Scale bar = 50 μm. Cul4a: cullin 4a.

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  Figure 5. PARP1 was a novel Cul4a-interacting protein, and the interaction was enhanced under oxidative stress. (a) Coimmunoprecipitation (co-IP) and western blotting (IP-western) using anti-PARP1 antibody or negative control IgG and Protein A/G immunoprecipitation magnetic beads followed by anti-Cul4a western blot were performed to identify endogenous interaction between PARP1 and Cul4a. (b) Anti-Flag antibody and Protein A/G immunoprecipitation magnetic beads followed by anti-PARP1 western blot were performed to identify semiexogenous interaction between Flag-Cul4a and PARP1. (c) Endogenous interaction between PARP1 and Cul4a was enhanced by treatment of 200 μM H2O2 for 2 h, which were evaluated by coimmunoprecipitation using anti-PARP1 antibody. (d) Semiexogenous interaction between Flag-Cul4a and PARP1 was enhanced by treatment of 200 μM H2O2 for 2 h, which were evaluated by coimmunoprecipitation using anti-Flag antibody. GAPDH was used as a loading control. Cul4a: cullin 4a; PARP1: poly (ADP-ribose) polymerase-1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

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  Figure 6. Cul4a can decrease the expression of PARP1. (a) Cul4a gradient overexpress with different amounts of plasmid in H9c2 cells, the expression of PARP1 decreased. (b) Knockdown Cul4a with control siRNA and Cul4a siRNA target sequence 1, 2, and 3, the expression of PARP1 with Cul4a siRNA target sequence 1 and 2 increased. α-Tubulin was used as a loading control. Cul4a: cullin 4a; PARP1: poly (ADP-ribose) polymerase-1.