Extended Data Fig. 6: Type I IFN signaling in Er1^Lyz2/− BMDMs and in vivo antibody blockade experiments.

(A.) Immunofluorescence detection of the colocalized DNA damage markers γ-H2A.X and 53BP1 in wt untreated and wt etoposide-treated BMDMs. The percentage of positive cells for over 2 colocalized γ-H2A.X⁺53BP1⁺ foci/cell is plotted. (At least 4 independent optical fields were counted from n = 3 biological replicates, pval = 0.0018) Single-channel images of Extended Data Fig. 6A are shown in Supplementary file 3A(B.) Quantitation of the mRNA levels of chemokine genes in Er1^Lyz2/− BMDMs, as shown. The mRNA levels of these genes in wt BMDMs are indicated with the red dotted line (n = 3-6, exact pvalue provided in Source Data file). (C.) (Top) Western blotting for the detection of interferon beta (IFN-β) protein levels secreted in the supernatants of wt and Er1^Lyz2/− BMDMs. The splice point is indicated by a vertical black line. Equal volumes of concentrated supernatants were loaded on the gel. For the normalization of the secreted protein levels, equal volumes of cells lysed with RIPA buffer were loaded and the membranes were probed with beta tubulin. (Bottom) Blots for the quantification of interferon regulatory factor 5 (IRF5) and phosphorylated signal transducer and activator of transcription 1 (pSTAT1) versus total STAT1 protein levels. Actin was used for normalization (n = 3-4, pval = 0.0011 for IFN-β, pval= 0.009 for STAT1, pval = 0.048948 for IRF5). (D.) Western blot analysis of ERCC1 protein levels in CD4⁺ T cells purified from wt or Er1^Lyz2/− mice at the age of 10 months (n = 3). (E.) Gating strategy for the flow cytometry analysis of CD4⁺ T cells in a BMDM-CD4⁺ T-cell co-culture. (F-G.) Flow cytometry analysis of splenocytes isolated from wt and Er1^Lyz2/− anti-MHC-II-treated mice and from wt and Er1^Lyz2/− isotype control-treated mice and stained for (F.) the CD44 and CD62L activation T-cell markers and (G.) the CD11b myeloid cell marker and MHC-II. MHC-II levels exhibit a drop in the CD11b⁺ population (n = 3, pval = 0,0384). (H.) Immunofluorescence analysis of C3 complement protein localized in the kidney glomeruli of Er1^Lyz2/− isotype control-treated and Er1^Lyz2/− anti-MHC-II-treated animals. Representative images and plots with the MFI quantification of C3 are shown (n = 3, pval = 0.0343). (I.) H&E staining of kidneys derived from Er1^Lyz2/− anti-CD4-and isotype control-treated animals. The arrows point to inflammatory foci (n = 6-8, pval = 0,0452). Error bars indicate S.E.M. among replicates. Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01, ***≤0.01 (two-tailed Student’s t-test). Scale bars: 10μm.

Source data