simple chip enzymatic chromatin ip kit  (Cell Signaling Technology Inc)

Journal: The FASEB Journal

Article Title: ATF7 – PINK1 Axis Governs Mitophagy and Intestinal Inflammation in Ulcerative Colitis

doi: 10.1096/fj.202500813R

Figure Lengend Snippet: ATF7 regulates PINK1 expression and mitochondrial integrity in colonic epithelial cells. (A) Heatmap showing ChIP‐seq read distribution around transcription start sites (TSS) in colonic mucosal samples from UC patients. (B) Genomic annotation of enriched ChIP‐seq peaks indicates significant localization at TSS regions. (C) KEGG pathway enrichment analysis of genes associated with ChIP‐seq peaks reveals multiple pathways, with mitophagy displaying the highest rich factor. (D) Visualization of ChIP‐seq reads at the PINK1 transcription start site (TSS) using Integrative Genomics Viewer (IGV), highlighting ATF7 enrichment. (E) Transmission electron microscopy images of colonic epithelial cells from wild‐type (WT) mice and ATF7‐deficient ( ATF7 −/− ) mice, with or without DSS‐induced colitis. Damaged mitochondria, characterized by swelling, were more prevalent in the ATF7 −/− + DSS group. Scale bar: 1 μm. (F) Quantification of mitochondrial length, indicating a reduction in the ATF7 −/− + DSS group. Data are presented as mean ± SD. Statistical significance was determined using one‐way ANOVA ( n = 3–6, p < 0.05; ns, not significant).

Article Snippet: Chromatin immunoprecipitation assays were conducted using the Simple ChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology, Cat# 9004, MA, USA) along with anti‐ATF7 antibody (Abcam, Cat# ab183507, Cambridge, UK).

Techniques: Expressing, ChIP-sequencing, Transmission Assay, Electron Microscopy

Journal: The FASEB Journal

Article Title: ATF7 – PINK1 Axis Governs Mitophagy and Intestinal Inflammation in Ulcerative Colitis

doi: 10.1096/fj.202500813R

Figure Lengend Snippet: ATF7 regulates PINK1‐mediated mitophagy in intestinal epithelial cells. (A) ChIP assay showing ATF7 enrichment at the PINK1 promoter in CCD 841 CoN cells. Data are presented as ChIP/Input ratios. (B) Quantitative PCR confirming ATF7 knockdown in CCD 841 CoN cells transfected with sgRNA targeting ATF7 (sgATF7) compared to control sgRNA (sgCon). (C) Dual‐luciferase reporter assay showing reduced luciferase activity in cells with ATF7 knockdown (sgATF7) using the wild‐type (WT) PINK1 promoter construct. Luciferase activity was unchanged in cells with a mutated (Mut) promoter construct. (D) Representative immunofluorescence images of CCD 841 CoN cells under the indicated conditions. Top row: Co‐localization of PARKIN (green) and PINK1 (red). Middle row: MitoTracker (red) and LC3B (green) co‐localization indicating autophagosome formation. Bottom row: Co‐localization of MitoTracker (green) and LysoTracker (red) reflecting mitolysosome formation. (E) Live‐cell imaging using the mito‐Keima probe to assess mitophagy flux. Red fluorescence (acidic environment) indicates mitolysosome formation, while green fluorescence marks mitochondria in neutral pH. (F) Quantification of red‐to‐green fluorescence ratio from mito‐Keima analysis. Data represent mean ± SD ( n = 3–6); statistical analysis was performed using one‐way ANOVA. * p < 0.05; ns, not significant.

Article Snippet: Chromatin immunoprecipitation assays were conducted using the Simple ChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology, Cat# 9004, MA, USA) along with anti‐ATF7 antibody (Abcam, Cat# ab183507, Cambridge, UK).

Techniques: Real-time Polymerase Chain Reaction, Knockdown, Transfection, Control, Luciferase, Reporter Assay, Activity Assay, Construct, Immunofluorescence, Live Cell Imaging, Fluorescence