Fig 2: MAM-localized Rac1 inhibits RLR signaling in a protein geranylgeranylation–dependent manner.(A to C) Quantitative RT-PCR analysis of transcript abundance of Ifnb1, Il6, Rac1, and Rac2 in wild-type (WT), Rac1−/−, or Rac2−/− iBMDMs 4 hours after SeV infection. (D) Immunoblot of phospho-IRF3 and phospho-IKK in cell lysates of wild-type or Rac1−/− iBMDMs following SeV infection. Numbers below lanes indicate densitometry of phosphorylated IRF3 or IKK relative to that of total IRF3 or IKK, respectively. (E to H) Quantitative RT-PCR analysis of transcript abundance of Ifnb1, Il6, and SeV-P (E to G) and immunoblot of Rac1 (H) in Rac1−/− iBMDMs reconstituted with wild-type, C189S, G12V, or G12V/C189S of Rac1 4 hours after SeV infection. (I and J) Confocal microscopy of immunofluorescent staining of Rac1 (green) and SIGMA1R (red) in Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDMs 4 hours after SeV infection. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 10 μm (I). See each single channel in fig. S3J. The percentage of colocalization between Rac1 and SIGMA1R was calculated using the ImageJ software from at least 50 cells randomly selected from the immunostaining slides (J). (K to N) Representative PLA images of Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDMs (K) or Rac1−/− iBMDMs reconstituted with wild-type, G12V, or C189S of Rac1 (M) 4 hours after SeV infection. The cells were probed with Rac1 and SIGMA1R antibodies. Red spots indicate the association of Rac1 with SIGMA1R. (K and M) Cells were counterstained with DAPI (blue). Scale bars, 10 μm. (L and N) Quantitation of PLA spots per cell in (K) and (M) (n = 20 cells). (O) Schematic display of the C-terminal amino acid sequence of Rac1 highlighting the C178 for palmitoylation and C189 for geranylgeranylation sites. (P) Immunoblots of Rac1 after S-palmitoylated protein assay (acyl-RAC; see Materials and Methods) of captured proteins (Pulldown) and inputs from Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDM cell lysate following infection with or without SeV for 4 hours. (Q) Quantitative RT-PCR of Ifnb1 and Il6 abundance in MEF cells stably expressing wild-type and C178S mutant forms of Rac1 after transfection with poly(I:C) LMW. **P < 0.01, ****P < 0.0001 (two-way ANOVA). Data are representative of three independent experiments.

Fig 3: Apoptosis inhibits cytokine production.A, B Quantitative RT-PCR analysis of CXCL10 and MCP1 mRNAs in 293 T cells or 293-TLR3 cells treated with Etop, TG, or ST in the absence or presence of IL-1β (10 ng/mL) for 4 hr (A), TNFα (10 ng/ml) for 2 h (B). C, D Quantitative RT-PCR analysis of Cxcl10 and Ifnb1 mRNA in macrophages from WT mice treated with Etop, TG and ST, and infected with HSV or SeV for 8 h. E Quantitative RT-PCR analysis of ifnb1, cxcl10 mRNA in peritoneal macrophages from WT mice treated with Etoposide (Etop, 100 μM, 24 h), Thapsigargin (TG, 2 μM, 24 h) and Staurosporine (ST, 1 μM, 8 h), then treated with poly(I:C) (2 μg/ml, 2 h). F, G Sex- and age-matched mice (n = 10) were ip injected of 20 mg/kg doxorubicin (DOX or Doxo) or PBS for 5 days, ip injected with poly(I:C) or PBS for 2 h. Cxcl10 and il-6 mRNAs of heart were analyzed by qRT-PCR (F), serum concentrations of the indicated cytokines were measured by ELISA (G). Data are representative of three independent experiments (means with SEMs). *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig 4: Anthracyclines affect RelA subcellular localization and compromise RelA binding to NFKBIA and TNF promoters, but not to IFNB1 promoter.Reporter assays suggest that the RelA domain targeted by Epirubicin is the REL-homology domain (RHD). (A) RelA immunolocalization in macrophages challenged with E. coli for 4 hr and left untreated (control) or treated with 2 µM of Epirubicin (Epi); scale bar = 20 µm. (B) I?Ba degradation kinetics in macrophages following E. coli challenge in the absence or presence of 2 µM of Epi for 1 hr at the time of E. coli challenge and using total RelA levels as loading control. (C) Macrophages were either left untreated (top panel) or treated with Epi (bottom panel) and challenged with E. coli for the indicated times in the presence of the proteasome inhibitor MG132 (10 µM) or its vehicle DMSO; I?Ba degradation was assessed using total RelA as loading control. (D) Macrophages were challenged with lipopolysaccharide (LPS), treated with 2 µM of Epi or Aclarubicin (Acla) and an anti-RelA antibody was used to immunoprecipitate the associated chromatin, from where the promoter sequences of NFKBIA, TNF, and IFNB1 were amplified. (E) HEK293 cells were challenged with TNF, treated with 2 µM of Epi or Acla and an anti-PolII antibody was used to immunoprecipitate the associated chromatin, from where the promoter sequence of NFKBIA promoter was amplified. (F) HEK293 cells were transiently transfected with a ?B-luciferase reporter alone or in conjunction with full-length RelA or RelA (2-320)-VP16 or TET-RelA (268-551) and cells were left untreated or treated with 2 µM of Epi for 16 hr. A–C show one representative experiment using macrophages from at least three independent animals tested; D and E show arithmetic means and standard deviations of technical replicates from one representative experiment of at least four independently performed assays; F shows arithmetic means and standard deviations of technical replicates from one representative experiment of two independently performed assays. *p < 0.05; **p < 0.01; ***p < 0.001. Figure 3—source data 1.Western blot in Figure 3B. Figure 3—source data 2.Original blot of I?Ba in Figure 3B. Figure 3—source data 3.Original blot of total RelA in Figure 3B. Figure 3—source data 4.Western blot in Figure 3C. Figure 3—source data 5.Original blot of I?Ba in Figure 3C, no Epirubicin (upper panel, no Epi). Figure 3—source data 6.Original blot of I?Ba in Figure 3C, with Epirubicin (lower panel, Epi). Figure 3—source data 7.Original blots of total RelA in Figure 3C (both panels).

Fig 5: RIPK1 mediates hyperactivation of type I IFN signaling in the absence of caspase-8(A and B) Immunoblots of pSTAT1 and tSTAT1 in untreated BMDMs from indicated genotypes.(C) qRT-PCR analysis of Ifnb1, Zbp1, and Cxcl10 gene expression from untreated BMDMs of indicated genotypes.(D and E) Gene expression profiling of microarray results from untreated WT, Casp8-/-Ripk3-/-, Ripk3-/-, and Casp8-/-Ripk3-/-Ripk1-/- BMDMs. The heatmap of differentially expressed ISGs in the REACTOME interferon alpha beta signaling pathway (D, left), the heatmap of differentially expressed cytokine and cytokine receptor interaction genes in the KEGG pathway (D, right), and the enrichment plot of the REACTOME interferon alpha beta signaling pathway comparing Casp8-/-Ripk3-/- and Casp8-/-Ripk3-/-Ripk1-/- BMDMs (E) are shown.(F) Immunoblots of phosphorylated RIPK1 (pRIPK1 [S166]) and total RIPK1 (tRIPK1) in BMDMs of indicated genotypes. Full-length and cleaved tRIPK1 bands are shown.(G) Immunoblots of pSTAT1 and tSTAT1 in BMDMs from indicated genotypes left untreated, treated with Nec-1s (50 µM), or treated with Nec-1s (50 µM) and IFNß (100 ng/mL) for 6 h.(H) IFNß concentration in the culture supernatant from BMDMs of indicated genotypes left untreated or treated with Nec-1s (50 µM, overnight).Mean + SEM shown (C, H). ß-Actin was used as a loading control. Data are representative of at least two (F) or three independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001 (one-way ANOVA). Molecular weight markers are indicated on the right side of blots.