Extended Data Fig. 3: Senescent macrophages are characterized by elevated interferon signaling and have augmented responses towards LPS.
(a) Venn-diagram analysis of all upregulated genes across irradiation and doxorubicin-induced senescence models. (b)Volcano plots are differentially expressed (log2FC > 1.3 and FDR < 0.05) between control and senescent Sen(Doxo) macrophages. (c) Kegg terms associated with the genes up/down-regulated by both Sen(Doxo) macrophages compared to control macrophages. (d) Volcano plot of upregulated (red) and downregulated (blue) proteins by FDR adj p-value and fold change from SASP-proteomics (SASPomics) mass-spectrometry between control and Sen(Doxo) macrophages. (e) Kegg terms associated with the proteins up-regulated by Sen(Doxo) over control macrophages. (f) Venn-diagram analysis of all upregulated SASP proteins across irradiation and doxorubicin-induced senescence models. (g) Relative mRNA transcript levels of Cmpk2 across passage control, Sen(IR) and Sen(Doxo) macrophages. Asterisks represent the p-value of ANOVA ≥ 0.05 (ns), <0.05 (✱), <0.01 (✱✱), <0.001 (✱✱✱), <0.0001 (✱✱✱✱). (h) Relative mRNA transcript levels of M1 and senescent genes in response to LPS (24 hr 100 ng per mL) across P.Control, Sen(IR) and Sen(Doxo) macrophages. Asterisks represent the p-value of ANOVA ≥ 0.05 (ns), <0.05 (✱), <0.01 (✱✱), <0.001 (✱✱✱), <0.0001 (✱✱✱✱). (i) Phagocytosis assay using GFP-labeled E.Coli slurry. Images were taken at 40x magnification. Quantification of percent cells was performed using Image J software using 16-bit images. (j) Efferocytosis assay using apoptotic bodies derived from EdU-labeled MC38 cancer cells. Mean, +/− SE, EdU+ macrophages. N = 3 biological replicates, p-value from students t-test. (k) Relative mRNA transcript levels of M2 genes in response to IL-4 (24 hr 10 ng per mL) markers across P.Control, Sen(IR) and Sen(Doxo) macrophages. Asterisks represent the p-value of ANOVA ≥ 0.05 (ns), <0.05 (✱), <0.01 (✱✱), <0.001 (✱✱✱), <0.0001 (✱✱✱✱). (l) Arginase activity in cellular lysates as assessed by urea production. Asterisks represent the p-value of post-hoc 2-way ANOVA ≥ 0.05 (ns), <0.05 (✱), <0.01 (✱✱), <0.001 (✱✱✱), <0.0001 (✱✱✱✱).
