Figure 2

Enrichment of inflammatory genes in the transcriptome of senescent macrophages. (A) Volcano plot of the RNA sequencing (RNAseq) results comparing senescent and control macrophages. RNAseq was performed on 3 independent samples of both control and senescent macrophages. Upregulated and downregulated genes in senescent macrophages are reported as red dots and blue dots, respectively. Gray dots represent unregulated genes. Gene expression was considered regulated when its transcript levels changed two-fold, with a p-value ≤ 0.01 (p-adj-value ≤ 0.25). (B) DiRE analysis obtained from top 95 most significantly upregulated genes in senescent macrophages according to RNAseq results, showing the 10 most important transcription factors (DiRE: distant regulatory elements of co-regulated genes). Occurrence refers to the fraction of regulatory elements containing the binding site for the indicated transcription factor. (C) Hallmark signatures found by analyzing the regulated genes from RNAseq results with the Gene Set Enrichment Analysis (GSEA) software version MSigDB 2023.2. Sen MΦ: senescent macrophages, Ctrl MΦ: control macrophages, NES: normalized enrichment score. (D) Heatmap indicating upregulated cytokines in Sen MΦ from RNAseq results. (E) QPCR validation of upregulated transcripts in Sen MΦ found in A. The dotted line represents RNA levels of Ctrl MΦ. RNA levels were normalized over Tbp and β-Actin. The experiment was performed 3 times. Error bars represent standard deviation; * p-value < 0.05; ** p-value < 0.01 using Student’s t-test.