Fig. 2
From: C1Q+ TPP1+ macrophages promote colon cancer progression through SETD8-driven p53 methylation
p53K382me1 expression is specifically enriched in CD68+ macrophages present in IBD and CRC patients. A Immunofluorescence analysis of p53K382me1 and CD68 in tumor tissue derived from a p53WT CRC patient. Nuclei were counterstained by Toto-3. Scale bars, 40 µm. B p53K382me1 positivity percentage of CD68+ (macrophages), CD163+ (TAMs), CD8+ (lymphocytes), CD4+ (lymphocytes), CD15+ (B cells) and CD20+ (neutrophils) immune cells in tumor tissues derived from p53WT CRC patients. Data represent mean ± SD of three independent experiments. ns, not significant. C Analysis of KMT5A (SETD8) and CD68 relative mRNA expression levels in normal and inflamed tissues derived from IBD patients in E-MTAB-184 array express dataset. D Immunofluorescence analysis of p53K382me1 and CD68 in inflamed and peri-inflamed tissues derived from patients affected by Crohn’s disease (CD) and Ulcerative colitis (UC). Nuclei were counterstained by Toto-3. Scale bars, 40 µm. E p53K382me1 positivity percentage of total cell population (upper panel) and CD68+ macrophages (lower panel) in IBD patients as in (D). Data are expressed as mean ± SD of three independent experiments. F Immunohistochemical analysis of p53K382me1 on peri-inflamed tissue of a Crohn’s disease (CD) patient as measured by Image J software. Scale bars, 40 µm (left panels). Scheme showing the basal, transit-amplifying cells (TAC) and apical compartments of a colon crypt (right upper panel). p53K382me1 positivity percentage of epithelial cells in the indicated colon crypt compartments as shown in the left panels. Data are expressed as mean ± SD of three independent experiments. ns, not significant (right lower panel). G Scheme showing the generation of three conditioned media (CM) and its administration on healthy colon cells, CR-CSphCs and macrophages. Immunoblot analysis showing the expression of the indicated proteins in healthy colon cells, CR-CSphCs#14 and THP1 treated with three CM for 72 h. H Heatmap of the p53 pathway-related genes (2−ΔCt expression values) in healthy colon cells and CR-CSphCs#14 treated with CM3 for 48 h. Data are presented as normalized mRNA expression values of 2 biological replicates. The color key represents the normalized expression values: blue (low) to red (high). I The top 15 differentially expressed canonical pathways in CR-CSphCs#14 treated as in (H) obtained by Enrichment Pathway Analysis based on the Reactome Gene set (Molecular Signature Database, MSigDB), utilizing msigdbR library and p < 0.05. Pathways related to p53 signaling are among the top differentially expressed pathways. J Lollipop plot representing the number of cytokines released by CR-CSphCs#8 and THP1 cells (CM3) compared with CM1 as measured by Luminex Bio-plex assay. Data are expressed as LogFC values of 2 biological replicates. K Analysis of IL-6 and CCL2 (MCP-1) relative mRNA expression levels in normal and tumor tissues derived from CRC patients in TNM plot dataset. See also Figure S2