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Fig. 5 | Cell Division

Fig. 5

From: A positive feedback loop between EZH2 and NOX4 regulates nucleus pulposus cell senescence in age-related intervertebral disc degeneration

Fig. 5

Regulation of NOX4 expression by EZH2. a RT-qPCR for NOX4 in NP cells with EZH2 interference (siEZH2) and GSK126 treatment (5 μM for 4 or 8 days). NP cells transfected with scrambled siRNA control (siCtrl) were used as a control for siEZH2. b Reactive oxygen species (ROS) levels measured using the DCFH-DA ROS-sensitive dye and flow cytometry. c Immunoblot analysis for EZH2, H3K27me3, H3K27ac, and NOX4 in NP cells overexpressing EZH2, treated with GSK126 or siEZH2 cells. β-actin and H3 were used as loading controls. NP cells transfected with empty lentivirus vectors were used as controls for groups overexpressing EZH2. DMSO was used as a control for the inhibitor. d OD analysis of the immunoblot analysis results in c. The data are represented as the mean ± SEM (n = 3). e, f ChIP-qPCR for H3K27me3 and H3K27ac at the proximal promoter regions of the NOX4 gene in cultured rat NP cells. Cells were treated with DMSO and GSK126 (5 μM) for 8 days, and primers were chosen for NOX4 constructive enhancer regions. Rabbit IgG was used as an IP control. The data are represented as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01. pm 1 primer 1, pm 2 primer 2, pm 3 primer 3

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