Fig 2: Ripk3 transcripts are present in atherosclerotic plaques at very low copy numbers. (A-D) Sequential cross sections of control aortic roots were immunostained for the endothelial cell marker CD31 (green) and macrophage marker CD68 (red) (A,C) or for the smooth muscle cell marker aSMA (red) (B,D) and were co-stained to identify nuclei (Hoechst; blue). Two plaque regions that contain all three cell types are shown. (E-N) RNAScope® RNA in situ hybridization was performed on sequential sections of the two separate regions of control aortic roots shown in A-D. Ripk3 probe was used to identify Ripk3 transcripts (pink) and co-stained with DAPI (blue) to identify nuclei (E,G). F and H show magnifications of the boxed regions (without DAPI) in E and G, respectively. Positive control probes Ubc (pink; high transcript copy number) and Polr2a (red; low transcript copy number) were used to show the efficacy of the assay (I,L). J,K and M,N show magnifications of the boxed regions (without DAPI) in I and L, respectively. Positive signal is determined by the presence of ‘dots’ which are each meant to represent a single transcript. Images are representative of in situ hybridization experiments performed on sections from three separate control mice (n=3). Scale bars: 25 µm.

Fig 3: Bone-marrow-derived macrophages exhibit significant reduction in RIPK3 levels in Ripk3ΔGlobal, Ripk3ΔMΦ-Cre and Ripk3ΔEC-Cre mice. (A,B) BMDMs were isolated, differentiated on chamber slides for 7 days, immunostained for macrophage markers CD68 (red; A) and CD11b (pink; B), and co-stained for nuclei (Hoechst; blue). All cells from seven separate BMDM isolations were positive for these markers (n=7). (C) A phagocytosis assay was performed on BMDMs with fluorescent polystyrene beads. Phagocytic cells display TRITC+/Hoechst+ signal, and 85% of cells from two separate BMDM isolations were double positive (n=2). (D-F) Protein or RNA was collected from control, Ripk3ΔGlobal, Ripk3ΔMΦ-Cre and Ripk3ΔEC-Cre BMDMs. RNA was converted to cDNA and analyzed by qPCR for Ripk3 levels (D). Protein lysates were immunoblotted to identify CD11b, RIPK3 and β-actin (loading control) (E) and quantified (F). (G) RNA from control and Ripk3ΔGlobal BMDMs was converted to cDNA and analyzed by qPCR for Ripk3-Exon 10 levels. For panels D,F,G, each dot represents a BMDM isolation from an individual animal. Statistics for panels D and F were calculated using one-way ANOVA with Dunnett's multiple comparisons test. Overall ANOVA P-values (prior to the post hoc tests) are 0.0002 (D) and <0.0001 (F). Statistics for panel G were calculated using an unpaired t-test with Welch's correction. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data are mean±s.d. Scale bars: 25 µm.

Fig 4: RIPK3, MLKL and TRIF are required for RIPK1-independent necroptosis in Atg16l1-deficient BMDMs.(A-E) Immunoblot (A, C) and cell death assays (B, D, E) of BMDMs from mice of indicated genotypes treated with combinations of LPS/zVAD/Nec-1 following CRISPR-mediated deletion of RIPK3, MLKL or GSDMD (A, B) (n = 4) or TRIF (C–E) (n = 6). Cell death assayed by PI staining and live-cell imaging for 12–16 hr. Data in (A, B) are representative of three independent experiments; (C, D, E) are representative of four independent experiments. **p<0.01, ***p<0.001, ****p<0.0001. Bar graphs depict mean. NTC = non targeting gRNA.10.7554/eLife.44452.010Figure 2—source data 1.RIPK3, MLKL and TRIF are required for RIPK1-independent necroptosis in Atg16l1-deficient BMDMs.

Fig 5: Elevated ZBP1 in Atg16l1-deficient BMDMs suppresses TRIF-mediated necroptosis.(A) ZBP1 turnover in Atg16l1-WT and Atg16l1-cKO BMDMs following cycloheximide (CHX) treatment for indicated time points. Representative immunoblot (top), ZBP1 quantification by densitometry (bottom) normalized to ZBP1 band intensity in WT samples at 0 hr. (B, C) immunoblot (B) and cell death (C) assays of BMDMs from mice of indicated genotypes treated with combinations of LPS/zVAD/Nec-1 following CRISPR-mediated deletion of Zbp1, Ticam1 or both (n = 4). (D, E) cell death assayed in Atg16l1-WT or Atg16l1-cKO BMDMs following CRISPR-mediated Zbp1 deletion and a dose titration of LPS in the presence of 20 μM zVAD and/or 30 μM Nec-1 (n = 4). Dot-plots depict mean ±S.D. (F–H) immunoblots depicting accumulation of TRIF (F), autophosphorylated and total RIPK1 (G), autophosphorylated and total RIPK3 (H) in NP-40 insoluble lysates of BMDMs lacking both Atg16l1 and Zbp1 following induction of necroptosis via LPS/zVAD for 3 hr. Top panels represent short exposures; middle panels represent long exposures. *=non specific band. Data (A) are representative of four independent experiments, densitometry is pooled from four independent experiments. Data in (B, C) are representative of three independent experiments; (D–H) are representative of two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Bar graphs depict mean. NTC = non targeting control gRNA.10.7554/eLife.44452.025Figure 6—source data 1.Elevated ZBP1 in ATG16L1 deficient BMDMs suppresses TRIF-mediated necroptosis.