Fig 2: RA signaling was enhanced in Casp8−/− mouse embryos. a Immortalized MEFs derived from Wt (Casp8+/+) and Casp8−/− embryos were subjected to western blot analysis for Caspase-8 and RIPK3. b qRT-PCR analysis of RA-induced genes, Crabp2, Cyp26a1, and Rarb, was carried out in Casp8+/+ and Casp8−/− MEFs after treatment with or without 1 μM RA for 24 h. c Dual-luciferase reporter analysis of RARE was carried out using RA-treated Casp8+/+ and Casp8−/− MEFs. d qRT-PCR analysis of RA-inducible genes, Crabp2, Cyp26a1, and Rarb, was performed in E10.5 Wt (Casp8+/+) and Casp8−/− littermates (n = 10). RNA was extracted from whole embryos. e Expressions of RA-inducible genes, Crabp2, Cyp26a1, and Rarb were analyzed in E10.5 or E11.5 Wt (Casp8+/+) and Casp8−/− littermates by whole-mount in situ hybridization analysis. Scale bars, 2 mm. f, g Whole-mount in situ hybridization analysis of Rarb and Raldh2 was carried out using E11.5 Casp8−/− embryos (n = 5). Scale bars, 2 mm. Views of heart and AGM (f), and neural tube (g) were shown. Scale bars, 1 mm. h Whole-mount in situ hybridization analysis of Rarb was carried out using representative E11.5 embryos with the indicated genotypes (n = 5). Scale bars, 2 mm. i qRT-PCR analysis of RA-inducible genes, Crabp2, Cyp26a1, and Rarb, was carried out as described in d. **p < 0.01 and *p < 0.05

Fig 3: LeTx Triggers Caspase-8 Activation and Apoptosis in Caspase-1-Deficient Macrophages(A–D) B6Nlrp1b+ and B6Nlrp1b+C1−/−C11−/− BMDMs were treated with LeTx for 3 hr before supernatants were analyzed for LDH activity (A), bright-field images were acquired (B), and cells were stained for annexin V and PI followed by FACS analysis (C) and quanitification of annexin V-positive cells (D).(E and H) Wild-type and caspase-1/11-deficient BALB/c BMDMs were stimulated with LeTx for 3 hr and then microscopically analyzed (E), and lysates were immunoblotted for caspase-1 and caspase-8 (H).(F and G) BMDM lysates of the indicated genotypes were immunoblotted for RIPK3 and RIPK1 (F) and caspase- 1 and caspase-8 (G) following LeTx-treatment for the indicated durations.(I–K) z-IETD-fmk-pretreated B6Nlrp1b+C1−/−C11−/− BMDMs were stimulated with LeTx for 3 hr before lysates were immunoblotted for caspase-8 (I). Cells were analyzed by FACS (J) and annexin V-positive cells (K) were quantified in parallel.(L and M) BMDMs were incubated with TAT-CrmA for 10 min, followed by LeTx for 3 hr, before lysates were immunoblotted for caspase-1 and caspase-8 (L), and bright-field images were acquired (M).All scale bars are set at 20 μm. All data are representative of results from three independent experiments. Data are shown as mean ± SD from a single representative experiment of at least three independent experiments, with each condition performed in triplicate.

Fig 4: RIPK3‐MLKL axis does not impact the proliferation and death of DP thymocytes. A) Total thymocytes from Ripk3+/+ (n = 6) and Ripk3−/− (n = 6) mice were treated with TNF‐α (50 ng mL−1), z‐VAD (20 µm) and Smac mimetic (200 nm) for 5 and 24 h. Dead cells were identified by Annexin V and PI staining. B) The expression level of MLKL protein in primary immune cells. MLKL expression was measured by Western blot analysis. C) Representative histograms were the MLKL expression of the indicated thymic and splenic subsets. Expression levels of MLKL were measured by flow cytometry. Total thymocytes and splenocytes were treated with TNF‐α (50 ng mL−1), z‐VAD (20 µm) and Smac mimetic (200 nm) for the indicated time or without, and cells were analyzed by Annexin V and Propidium Iodide (PI) staining and measured by D) Flow Cytometry. RIPK3, MLKL and p‐MLKL protein was measured by E) Western blot analysis. Statistical analyses were performed using the two‐tailed unpaired Student t‐test. P values below 0.05 were considered significant in the following manner: *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: TCR‐mediated activation of LCK interacts with and RIPK3 to promote RIPK3 phosphorylation. A) Total thymocytes from Ripk3+/+ (n = 5) and Ripk3−/− (n = 5) mice were cultured with anti‐CD3 (1 µg mL−1) and anti‐CD28 (2 µg mL−1) or without for indicated time. Relative fluorescence of RIPK3 and p‐RIPK3 in DP T cells. B) Phosphorylation of RIPK3 increased in anti‐CD3 (1 µg mL−1) and anti‐CD28 (2 µg mL−1) stimulation. C) Experimental workflow for TAP (Tandem Affinity purification) pull‐down assay. An aliquot of each purified sample was loaded to SDS‐PAGE and stained with Coomassie brilliant blue. Potential RIPK3‐binding proteins were identified by LC‐MS/MS. Venn diagram represents the overlap of proteins and unique proteins identified by LC/MS/MS among TAP‐purified samples as indicated. Total 503 proteins were identified as specific RIPK3 binding proteins. There were several kinases and phosphatases. D) Computational docking model for RIPK3 (olive) and LCK (blue) predicted using ClusPro (see Materials and Methods). E) Western blot analysis after immunoprecipitation of mouse RIPK3 and mouse LCK in HEK293T cells. HEK293T cells were transfected with LCK and/or Flag‐RIPK3 expression constructs. Cells were harvested at 24 h after transfection. The endogenous RIPK3 interacted with LCK in response to anti‐CD3/CD28 stimulation. Interaction of RIPK3 and LCK was observed by F) Western blot analysis, and confirmed with G) Duolink proximity ligation assay. H) Tyrosine phosphorylation of RIPK3 were detected in LCK and Flag‐RIPK3 expressing HEK293T cells. Statistical analyses were performed using the two‐tailed unpaired Student t‐test. p values below 0.05 were considered significant in the following manner: *p < 0.05, **p < 0.01, ***p < 0.001.