Fig 1: The reduced sensitivity to TNF in Bcl-3-deficient mice is independent of lymphocytes.A Schematic showing that lethally irradiated WT and KO mice transplanted with wild-type bone marrow cells were injected intraperitoneally with T + D. B Survival of WT and KO recipients treated with T + D was monitored every 2 h for 2 days. C Schematic showing that lethally irradiated WT and KO mice transplanted with Bcl-3-deficient bone marrow were injected with T + D. D Survival of WT and KO recipients was monitored. E Liver tissues were collected and photographed at 4 h after injection. F H&E staining of livers of the recipients 4 h post injection (scale bar = 20 μm). G Immunohistochemical analysis of livers of the recipients were stained for cleaved Caspase 3 at 4 h after injection (scale bar = 100 μm). The integrated intensity is shown on the right. The results are shown as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2: MK2-Dependent Phosphorylation of RIPK1 at S321 Protects Cells from TNF-Induced Cell Death(A) Quantification of PI-positive WT and Ripk1S321D BMDMs treated with the indicated reagents for 5 hr.(B) DEVDase activity analysis of BMDMs treated with the indicated reagents for 1 hr.(C) Quantification of PI-positive primary WT and Ripk1S321D MEFs treated with the indicated reagents for 6 hr.(D) PLA of primary WT and Ripk1S321D MEFs using RIPK1 and caspase-8 antibodies. Cells were stimulated with the indicated reagents for 3 hr. The panel below shows quantifications of RIPK1/caspase-8 PLA speckles. Scale bar, 10 μm.Graphs show mean ± SEM, n = 3–8 independent repeats. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.See also Figure S6.
Fig 3: Ripk1K376R/K376R mutation increases RIPK1 kinase activity. a Immortalized Ripk1+/+, Ripk1K376R/K376R, and Ripk1−/− MEFs were treated for the indicated time with TNFα (20 ng/ml). The K63-ubiquitylated proteins were isolated by K63-TUBEs and analyzed by western blot. b TNF-RSC was immunoprecipitated using anti-Flag resin in Ripk1+/+ and Ripk1K376R/K376R immortalized MEFs treated for the indicated time with Flag-TNFα (100 ng/ml). The immunocomplexes were analyzed by western blot with indicated antibodies. c Nuclear extracts were collected from Ripk1+/+, Ripk1K376R/K376R, and Ripk1−/− immortalized MEFs treated with TNFα (20 ng/ml) at indicated times and analyzed by western blotting with antibodies against p65 and PCNA. d Ripk1+/+ and Ripk1K376R/K376R immortalized MEFs that stably expressed with the Flag-tagged IκBα-SR were stimulated with TNFα (40 ng/ml) for different periods of time. Cell death was measured by SytoxGreen positivity. e, f Complex II was immunoprecipitated using RIPK1 antibody (e) or RIPK3 antibody (f) in Ripk1+/+, Ripk1K376R/K376R, and Ripk1−/− immortalized MEFs treated for the indicated time with TSZ (e) or TZ (f). The immunocomplexes were analyzed by western blotting with indicated antibodies. T, TNFα (20 ng/ml); S, Smac mimetics (1 μM); Z, zVAD.fmk (1 μM). g Ripk1+/+ and Ripk1K376R/K376R immortalized MEFs that expressed with the Flag-tagged TAK1/TAB1 or TAK1-∆N were stimulated with TNFα (20 ng/ml) plus zVAD.fmk (10 μM) for different periods of time. The cell lysates were analyzed by western blotting with indicated antibodies. h Cell death of Ripk1+/+ and Ripk1K376R/K376R immortalized MEFs treated for 5 h with different stimulators was measured by SytoxGreen positivity. T: TNFα (20 ng/ml), Z: zVAD.fmk (10 μM), TAK1i: TAK1 inhibitor (500 μM), IKKi: IKK inhibitor(5 μM), MK2i: MK2 inhibitor (2 μM). In d, h, data are mean ± s.e.m. (n = 3 independent cell samples for each genotype). Statistical significance was determined using a two-tailed unpaired t test, n.s., P > 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001
Fig 4: Bcl-3 deficiency leads to decreased hepatoxicity and lethality in response to TNF in vivo.Ten- to 12-week-old male WT and Bcl-3 KO mice were injected intraperitoneally with T + D. A Survival was monitored every 2 h for 3 days. B Liver tissues were collected and photographed at 6 h after injection. C Levels of serum ALT (IU/L) in WT and KO mice treated with PBS or T + D. D Hepatic tissues were collected after injection and subjected to histological analysis using H&E staining (scale bar = 20 μm) and immunohistochemistry with anti-cleaved Caspase 3 antibody and TUNEL staining (scale bar = 100 μm). The black arrows denote hepatocytes with fragmented nuclei. The frequency of the integrated intensity of cleaved Caspase 3+ or TUNEL+ hepatocytes is shown in the graphs on the right. E Survival curve of WT and KO mice injected intravenously with ConA. F Liver tissues were collected and photographed at 13 h after injection. G Western blot analysis of cleaved Caspase 3 and PARP in liver tissues after ConA treatment for 13 h. The results are shown as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 5: RIPK1 K376R mutation cause embryonic lethality, while RIPK1 K115R mutation does not affect survival.a Numbers of offspring at different embryonic days and adult age from intercrossing Ripk1K376R/+ mice. b Representative embryos of the Ripk1K376R/+ intercross at E12.5. Scale bars on the top left of each image indicate 2 mm. c Yolk sac of Ripk1+/+, Ripk1K376R/+ and Ripk1K376R/K376R intercross labeled for cleaved caspase-3 [green] and PECAM-1 [red] [scale bar 50 μm]. d Hematoxylin and eosin staining [upper panels, scale bar 100 μm] and IHC for cleaved caspase-3 [lower panels, scale bar 200 μm] of Ripk1+/+ and Ripk1K376R/K376R embryos. e IHC labeling for cleaved caspase-3 in the placenta labyrinths of Ripk1+/+ and Ripk1K376R/K376R embryos [scale bar 100 μm]. f Numbers of offspring from intercrossing Ripk1K115R/+ mice. g Histology of liver, spleen and small intestines of 12–15 months aged Ripk1+/+ and Ripk1K115R/K115R mice [scale bar 100 μm liver and small intestine, 200 μm spleen]. h Serum cytokine levels of IL-6 in Ripk1+/+ [n = 5] and Ripk1K115R/K115R [n = 5] 12–15 months old. i Survival of Ripk1+/+ in black [n = 13 males] and Ripk1K115R/K115R in red [n = 13 males] mice in TNF-induced SIRS model with 500 μg/kg TNF injected iv. Difference between the two groups by Mantel–Cox test: p = 0.0095.
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