Journal: Cell Death Discovery
Article Title: BRD4 Inhibition alleviates sepsis-associated acute kidney injury via suppression of NOX4-mediated oxidative stress and inflammation
doi: 10.1038/s41420-026-03113-y
Figure Lengend Snippet: A Western blots. HK2 cells were subjected to LPS (500 ng/mL) for 24 h and then were assayed for P300 and BRD4. The lower panel is the quantification. Data were presented as means ± SD of three repeated cell assays. * P < 0.05, one-way ANOVA. B Western blots. Renal tissues from control (Ctrl) and LPS group mice were assayed for P300 and BRD4. The lower panel is the quantification. Data were presented as means ± SEM, n = 6, * P < 0.05, one-way ANOVA. C Co-IP assay. Renal tissues of control (Ctrl) and LPS group mice were tested for P300 and BRD4. The same tissue lysates were immunoprecipitated with isoform-matched immunoglobulin (IgG) or antibody to P300 and BRD4, and then immunoprecipitants were assessed for P300 and BRD4 by western blotting. D Quantification of Co-IP. Data were presented as means ± SEM based on three renal samples from each group. * P < 0.05, one-way ANOVA. E Western blots. HK2 cells were treated with LPS (500 ng/mL) in presence or absence of C646 (10 μM) for 24 h, and then the cell lysates were analyzed for P300 and NOX4 expression. Quantification represented densitometric analysis normalized to the corresponding loading controls from three independent experiments. Data were presented as means ± SD. * P < 0.05, two-way ANOVA. F HK2 cells were treated with LPS (500 ng/mL) in presence or absence of C646 (10 μM) for 24 h, and then the cell lysates were tested for H3K4ac, H3K14ac, H3K18ac, H3K27ac and H3K36ac. Total Histone 3 served as controls. G Quantifications of protein levels in (F). Data were presented as means ± SD of three repeated cell assays. * P < 0.05, two-way ANOVA. H, I ChIP assay. HK2 cells were treated with LPS (500 ng/mL) with or without JQ1 (10 μM) for 4 h. ChIP assay was performed with antibodies to BRD4, P300, acetylated histone H3 (H3ac) or isoform-matched IgG, respectively. The immunoprecipitated DNA were analyzed by regular PCR and qRT-PCR. The PCR products were analyzed on an agarose gel (left panel). qRT-PCR (right panel) was normalized to input DNA and presented as fold changes relative to Ctrl. Data were presented as means ± SD of three repeated cell assays. Data were presented as means ± SD of three repeated cell assays. * P < 0.05, one-way ANOVA.
Article Snippet: The kidney tissues lysates were first immunoprecipitated with antibody to BRD4 (E1Y1P, Cell Signaling Technology, USA) or P300 (D8Z4E, Cell Signaling Technology, USA), isoform-matched immunoglobulin (IgG, AC005, ABclonal, China) overnight and then incubated with Protein A + G Agarose (P2055, Beyotime, China) and washed.
Techniques: Western Blot, Control, Co-Immunoprecipitation Assay, Immunoprecipitation, Expressing, Quantitative RT-PCR, Agarose Gel Electrophoresis
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