Journal: Neural Regeneration Research

Article Title: Bromodomain-containing protein 4 knockdown promotes neuronal ferroptosis in a mouse model of subarachnoid hemorrhage

doi: 10.4103/NRR.NRR-D-24-00147

Figure Lengend Snippet: BRD4 knockdown inhibits RAF-1/ERK1/2 signaling following treatment with OxyHb. (A) Heatmaps of BRD4 binding peaks in HT-22 cells from the NC-Control and NC-OxyHb groups. The intensity of the color indicates the relative number of reads, and genes with similar distribution patterns were clustered together using a clustering algorithm to show trends in BRD4 binding to all genes. (B) Heatmap of BRD4 binding peaks in HT-22 cells from the NC and KD groups. The intensity of the color indicates the relative number of reads, and genes with similar distribution patterns were clustered together using a clustering algorithm to show the trends in BRD4 binding to all genes. (C) Bubble chart showing the top 10 GO terms of genes that exhibited more bound BRD4 in the NC-Control group compared with the NC + OxyHb group. (D) Bubble chart showing the top 10 gene ontology terms of genes that exhibited more bound BRD4 in the NC group than in the KD group. (E) KEGG analysis of genes that exhibited more bound BRD4 in the NC group than in the KD group. (F) Protein–protein interaction network of common genes involved in protein serine/threonine kinase activity that exhibited differential BRD4 binding among the three groups. The intensity of the color indicates the strength of the protein’s influence on other proteins in the network. (G) ChIP-qPCR analysis of BRD4 occupancy of the Raf-1 promoter. (H) Representative western blot image of Raf-1, p-ERK1/2, and ERK1/2 expression in HT-22 NC and KD cells treated with OxyHb. (I, J) Quantification of Raf-1 (I) and p-ERK1/2/ERK1/2 (J) expression levels ( n = 3 per group). Data are expressed as mean ± SD. * P < 0.05, *** P < 0.001 (unpaired t -test for G, or one-way analysis of variance followed by Tukey’s multiple comparison test for I and J). BRD4: Bromodomain-containing protein 4; ChIP-qPCR: chromatin immunoprecipitation-quantitative polymerase chain reaction; ERK1/2: extracellular-regulated kinase 1/2; GO: Gene Ontology; KD: knockdown; KEGG: Kyoto Encyclopedia of Genes and Genomes; NC: negative control; p-ERK1/2: phosphorylation-extracellular-regulated kinase 1/2; Raf-1: RAF proto-oncogene serine/threonine-protein kinase.

Article Snippet: Both the cells and cerebral cortex tissues were lysed in chilled radio immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China; Cat# P0013B) supplemented with 50× protease and phosphatase inhibitors (Beyotime, Cat# P1050).

Techniques: Knockdown, Binding Assay, Control, Activity Assay, ChIP-qPCR, Western Blot, Expressing, Comparison, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Phospho-proteomics