Volume 19, Issue 4, Pages 798-808 (April 2017) RIPK3 Mediates Necroptosis during Embryonic Development and Postnatal Inflammation in Fadd-Deficient Mice  Qun Zhao, XianJun Yu, HaiWei Zhang, YongBo Liu, XiXi Zhang, XiaoXia Wu, Qun Xie, Ming Li, Hao Ying, Haibing Zhang  Cell Reports  Volume 19, Issue 4, Pages 798-808 (April 2017) DOI: 10.1016/j.celrep.2017.04.011 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 19, 798-808DOI: (10.1016/j.celrep.2017.04.011) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Ripk3Δ/Δ Mice Suppress Necroptosis in Macrophages and MDFs (A) Targeting strategy for the introduction of the mutation in exon 3 of the Ripk3 locus with the CRISPR-Cas9 genome editing system. (B) Peritoneal macrophages from wild-type, Ripk3−/−, and Ripk3Δ/Δ mice were treated with DMSO (D), LPS (L, 100 ng/mL), zVAD (Z, 20 μM), TNF (T, 30 ng/mL), SmacM (S, 10 nM), and Nec-1 (N, 30 μM) as indicated for 24 hr. (C) BMDMs from the indicated genotypes were challenged with the indicated treatments as described in (B) for 24 hr. (D) Wild-type, Ripk3−/−, and Ripk3Δ/Δ MDFs were treated for 24 hr with DMSO, TNF (20 ng/mL), SmacM (1 μM), zVAD (20 μM), and Nec-1 (30 μM). Cell viability was determined by measuring ATP levels. Data are represented as mean ± SEM of triplicates. ∗∗∗p < 0.001. Cell Reports 2017 19, 798-808DOI: (10.1016/j.celrep.2017.04.011) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Ripk3Δ/Δ Mutant Is Defective in MLKL Processing (A) Auto-phosphorylation assay of wild-type RIPK3 or the RIPK3 mutants. Flag-tagged wild-type RIPK3 or the kinase-dead mutant (RIPK3K51A and RIPK3D161N) mice were transfected into 293T cells. After 24 hr, the cells were harvested, and phosphorylation of RIPK3 was analyzed by western blot analysis using anti-p-RIPK3. (B) Phosphorylation of MLKL in wild-type RIPK3 or the RIPK3 mutants. Flag-tagged wild-type or the kinase-dead mutant (RIPK3K51A and RIPK3D161N) and MLKL were co-transfected into 293T cells. After 24 hr, 20-μg aliquots of protein were subjected to western blot analysis to measure the levels of p-MLKL. (C) In vitro kinase assay. Flag-tagged wild-type RIPK3 or the kinase-dead mutant (RIPK3K51A and RIPK3D161N) were immunopurified and added into a kinase assay with ATP and the purified recombinant MLKL as described in the Experimental Procedures. The levels of p-MLKL were subjected to western blot analysis. (D) MDFs from wild-type, Ripk3−/−, and Ripk3Δ/Δ mice were treated with TNF (20 ng/mL) + SmacM (1 μM) + zVAD (20 μM) with or without Nec-1 (30 μM) for 6 hr. For reducing gels, the cells were harvested and then lysed with 1 × SDS sample buffer and boiled at 95°C for 5 min. Proteins were separated using reducing SDS-PAGE gels. For non-reducing gels, the cells were harvested and lysed with 1 × DTT-free sample buffer. Then, the samples were separated using non-reducing PAGE gels. The samples were analyzed by western blotting using antibodies as indicated. (E) Plasma membrane translocation of MLKL. MDFs from wild-type, Ripk3−/−, and Ripk3Δ/Δ mice were treated with TNF (20 ng/mL) + SmacM (1 μM) + zVAD (20 μM) with or without Nec-1 (30 μM). After 6 hr, the cells were harvested and solubilized in Triton X-114 lysis buffer and then separated into the aqueous phase (Aq) and detergent phase (Det), as described in the Experimental Procedures. β-actin is shown as the loading controls for soluble protein (Aq), while mitochondria inner membrane protein Cox4 is shown as the loading controls for membrane protein (Det). Cell Reports 2017 19, 798-808DOI: (10.1016/j.celrep.2017.04.011) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Cerulein-Induced Acute Pancreatitis and LPS/zVAD-Induced Necroptosis Are Impaired in Ripk3Δ/Δ Mice (A–C) Wild-type, Ripk3−/−, and Ripk3Δ/Δ mice (n = 5) were administered intraperitoneal injections of 50 μg/kg cerulein or saline every hour for 6 consecutive hours, and the mice were sacrificed at 24 hr after the first injection. The pancreas of each mouse was subjected to H&E staining and examined for cell death (A and B). (C) Blood was harvested at 24 hr after the last injection, and serum amylase activity was measured using an amylase assay kit. (D and E) Wild-type, Ripk3−/−, and Ripk3Δ/Δ mice intraperitoneally injected with vehicle or zVAD (20 mg/kg) 1 hr before intraperitoneal injections with PBS or LPS (10 mg/kg). Resident peritoneal cells were collected by lavage of the peritoneal cavity with 3 mL PBS at 24 hr after the injection. Mac-1+F4/80+ peritoneal macrophages were analyzed by flow cytometry (D) and quantification of results from (D) is shown in (E). Data are represented as mean ± SEM of triplicates. ∗p < 0.05, ∗∗∗p < 0.001. (F) Peritoneal cell viability was analyzed by trypan blue staining. Cell Reports 2017 19, 798-808DOI: (10.1016/j.celrep.2017.04.011) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Fadd−/−Ripk3Δ/Δ Mice Allow Normal Embryogenesis (A) Photographs of E11.5 embryos with the indicated genotype (n = 5). (B) Expected and observed frequencies of genotypes in offspring at weaning from the intercrosses of Fadd+/−Ripk3Δ/Δ and Fadd+/−Ripk3Δ/Δ mice. (C) MDFs from wild-type, Fadd−/−Ripk3−/−, and Fadd−/−Ripk3Δ/Δ mice were treated with DMSO (D), TNF (T, 20 ng/mL), SmacM (S, 1 μM), zVAD (Z, 20 μM), and Nec-1 (N, 30 μM) for 6 hr. Cell viability was determined by measuring ATP levels. Data are represented as the mean ± SEM of triplicates. ∗∗p < 0.01, ∗∗∗p < 0.001. (D) MDFs from wild-type, Fadd−/−Ripk3−/−, and Fadd−/−Ripk3Δ/Δ mice were treated with TNF (20 ng/mL) + SmacM (1 μM) + zVAD (20 μM) with or without Nec-1 (30 μM) for 6 hr. The cells were harvested and lysed with 1 × DTT-free sample buffer for non-reducing gel or with 1 × SDS sample buffer and boiled at 95°C for 5 min for reducing gel. Proteins were separated using reducing SDS-PAGE gels and detected by antibodies as indicated. (E) Plasma membrane translocation of MLKL. MDFs from wild-type, Fadd−/−Ripk3−/−, and Fadd−/−Ripk3Δ/Δ mice were treated with TNF (20 ng/mL) + SmacM (1 μM) + zVAD (20 μM) with or without Nec-1 (30 μM). After 6 hr, the cells were harvested and solubilized in Triton X-114 lysis buffer and then separated into aqueous phase (Aq) and detergent phase (Det), as described in the Experimental Procedures. Cell Reports 2017 19, 798-808DOI: (10.1016/j.celrep.2017.04.011) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Postnatal Lethality in the Fadd−/−Ripk3Δ/Δ Mice Is Due to Massive Intestinal Inflammation (A) Fadd+/−Ripk3Δ/Δ and Fadd−/−Ripk3−/− mice on postnatal days 1 and 2. (B) Genetic analysis of offspring from intercrossing of the Fadd+/−Ripk3Δ/Δ mice. Neonates were analyzed at birth and monitored for survival. (C) Survival of pups from the intercrosses of Fadd+/−Ripk3Δ/Δ mice. (D) Representative images of the intestines from wild-type, Fadd−/−Ripk3−/−, and Fadd−/−Ripk3Δ/Δ newborn mice. (E) Tissue sections from wild-type, Fadd−/−Ripk3−/−, and Fadd−/−Ripk3Δ/Δ mice were subjected to H&E staining. Cell Reports 2017 19, 798-808DOI: (10.1016/j.celrep.2017.04.011) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 Fadd−/−Ripk3Δ/Δ Mice Exhibit Increases in Inflammatory Cytokines Production (A) Graphs depicting real-time PCR analysis of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) expression levels in the intestines of Fadd−/−Ripk3Δ/Δ mice compared to wild-type and Fadd−/−Ripk3−/− mice. (B) Graphs depicting real-time PCR analysis of chemokine (Cxcl1, Ccl2, and Ccl5) expression levels in the intestines from mice with indicated genotypes. (C) ELISA for TNF-α, IL-1β, and IL-6 in the intestines of wild-type, Fadd−/−Ripk3−/−, and Fadd−/−Ripk3Δ/Δ mice (n = 5–6 mice per genotype). (D) Immunoblotting for RIPK1, RIPK3, and FADD in thymuses and spleens from Fadd−/−Ripk3Δ/Δ mice compared to wild-type mice. (E) Fadd−/−Ripk3−/− and Fadd−/−Ripk3Δ/Δ fetal liver macrophages were primed with LPS (100 ng/mL) for the indicated time points, and supernatants were collected and used for the detections of IL-1β by ELISA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Cell Reports 2017 19, 798-808DOI: (10.1016/j.celrep.2017.04.011) Copyright © 2017 The Author(s) Terms and Conditions