Fig 2: Cox2 controls the intrahepatic immune microenvironment during senescence surveillance(A) Ptgs2fl/fl mice underwent HDTV injection of transposons containing inactive NRASG12V / D38A or oncogenic NRASG12V to induce hepatocyte RIS with or without Cre recombinase before harvest at day 9 post-injection. Quantification of NRAS-positive area by IHC is shown. Dots indicate individual mice, and bars represent means. Data were analyzed by one-way ANOVA with Sidak’s multiple comparisons test; *p = 0.05.(B) t-distributed stochastic neighbor embedding (t-SNE) plots of multiplexed intrahepatic immune cell mass cytometry data from the same 18 mice in (A) (n = 6 per condition) using 23 metal-tagged antibodies against immunocyte surface markers (see Table S2).(B and C) Hyperspheres of immune cell phenotypes were identified by typical surface marker expression (B) and colored according to log-fold change in abundance (C) between NRASG12V versus NRASG12V + Cre conditions.(D and E) Live lineage-negative, non-granulocytic/non-DC immune cells were gated as in Figure S4C, before analysis of different monocyte populations based on CD68 and F4/80 expression, identifying monocytic immature myeloid cells (Mo iMCs) as CD68INT and F4/80LO; example dot-plots of monocyte populations from NRASG12V and NRASG12V + Cre-injected mice are shown in (D) with quantification in (E). Dots indicate individual mice, and bars represent means. Data were analyzed by one-way ANOVA with Sidak’s multiple comparisons test; **p = 0.01.(F) Histograms from CyTOF data demonstrating expression of Ly6C and CD11b on iMCs and macrophages (including Kupffer cells, CD68HI/F4/80HI) from NRASG12V + Cre-injected mouse liver.(G) Histograms from flow cytometry demonstrating expression of Ccr2 and Cxcr2 on iMCs from NRASG12V and NRASG12V + Cre-injected mouse livers.(H and I) Expression of Ly6C in the liver by IHC in same mice as in (A); example fluorescence IHC from NRASG12V + Cre-injected mouse demonstrating Ly6C+ cells surrounding an NRAS-positive hepatocyte in (H). Scale bar, 20 µm. (I) Quantification of Ly6C+ cells. Dots indicate individual mice, and bars represent means. Data were analyzed by one-way ANOVA with Sidak’s multiple comparisons test; **p = 0.01.See also Figure S4.

Fig 3: Effect of CRID3 on M1 and M2 cells in the injured spinal cord as distinguished by FCM. a Representative images of FCM in the spinal cords in sham, SCI (vehicle), and SCI (CRID3) groups. In the CD11b/SSC pseudocolor plots, the same size “region” of the nucleated CD11b+ cells was set for each sample, and then analyzed the proportion of each cell population in CD68/CCR7 and CD45/CD11b pseudocolor plots, respectively. In the CD45/CD11b pseudocolor plots, the “regions” of the CD11b+CD45−/low cells (R1) and CD11b+CD45high cells (R2) for each sample were further analyzed for the proportion of each cell population in CD68/CCR7. b Quantitative analysis of the indicated cells in the indicated groups. Data represent the mean ± SD (n = 6). **P < 0.01 (non-parametric Kruskal-Wallis ANOVA, followed by individual Mann-Whitney U tests)

Fig 4: Effect of CRID3 on M1 and M2 cells in the injured spinal cord as distinguished by IHF. (a-f) Representative images of CD68 (green) and CCR7 (red) (a-c) or Arg1 (e-f) expression in the spinal cords in sham, SCI (vehicle), and SCI (CRID3) groups. Cells were counterstained with Hoechst 33342 (blue) to visualize nuclei. (g and h) Quantitative analysis of CD68+CCR7+ (g) and CD68+Arg1+ (h) cells in the indicated groups. Data represent the mean ± SD (n = 6). *P < 0.05, **P < 0.01 (non-parametric Kruskal-Wallis ANOVA, followed by individual Mann-Whitney U tests)

Fig 5: Effect of CRID3 on the number and status of microglia and infiltrated macrophages in the injured spinal cord as distinguished by IHF. (a-f) Representative images of CD11b (red) and CD68 (green) (a-c) or CD45 (e-f) expression in the spinal cords in sham, SCI (vehicle), and SCI (CRID3) groups. Cells were counterstained with Hoechst 33342 (blue) to visualize nuclei. (g-k) Quantitative analysis of CD45+ (g), CD68+ (h), CD11b+ (i), CD68+CD11b+ (j), and CD45+CD11b+ (k) cells in the indicated groups. Data represent the mean ± SD (n = 6). *P < 0.05, **P < 0.01 (non-parametric Kruskal-Wallis ANOVA, followed by individual Mann-Whitney U tests)