Fig 1: ATR and PARP inhibition increases unrepaired damage and suppresses HR. (A) U87 WT and IDH1-R132H/+ cells were treated with olaparib (10 μM), AZD6738 (5 nM) or both for 4 and 24 h. Cell were fixed and stained for RPA70 and counterstained with DAPI. Cells with more than 20 RPA70 foci were counted in seven distinct fields. In total 250–350 cells were analyzed. Representative images of cells stained with RPA (green) and counterstained with DAPI (Blue) are shown. The images shown were acquired using a 40× objective lens. The scale bar is 10 μm. (B) HCT116 WT and IDH1-R132H/+ cells were treated with olaparib (1 μM), AZD6738 (500 nM) or both for 24 h. Cell were fixed and stained for γ-H2AX and counterstained with DAPI. Cells with more than 10 γ-H2AX foci were counted in eight distinct fields. In total 800–1000 cells were analyzed. Representative images of cells stained with γ-H2AX (green) and counterstained with DAPI (Blue) are shown. The images shown were acquired using a 40× objective lens. The scale bar is 10 μm. (C) Quantitation of ligand inducible U2OS-DR-GFP assay where cells were cultured with or without 300 μM 2HG for 3 days and with and without olaparib (1 μM), AZD6738 (500 nM) or both for 24 h prior to ligand induction (n = 3). (D) Quantitation of control U2OS-DR-GFP assay without ligand induction. Dotplots show GFP population with and without ligand addition. Error bars represent means ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05.
Fig 2: Expression of RPA1 during early limb development.(A) Uniform manifold approximation and projection (UMAP) plot of mouse embryonic limb mesenchymal cells. (B) Violin plot showing the DDR score and cell cycle score of mouse embryonic limb mesenchymal cells from embryonic day 9.5 (E9.5) to E18. (C) Heatmap illustrating the expression levels of representative DDR genes in mouse embryonic limb mesenchymal cells from E9.5 to E18.5. (D) Violin plot showing the expression levels of Rpa1 in mouse embryonic limb mesenchymal cells from E9.5 to E18.5. (E) Violin plot showing the expression levels of RPA1 in human embryonic limb mesenchymal cells from Pcw5.1 to Pcw9.3. (F) Immunofluorescence staining on murine limbs from E11.5 to E13.5 (n = 3, and representative images are shown). DAPI, 4′,6-diamidino-2-phenylindole.
Fig 3: ZBP1 mediates PANoptosis induced by Rpa1 depletion.(A and B) Western blot analysis of ZBP1 and RPA1 in limb bud lysates from E12.5 WT and CKO embryo (A) and in siRpa1 cells (B). Asterisk denotes a nonspecific band (n = 3, and representative images are shown). (C) Representative images of cell death in cells treated with siNC, siRpa1, and the combination of siRpa1 and siZbp1. Scale bar, 100 μm. (D) Quantification of cell death in (C) (n = 3). One-way ANOVA. (E) Immunoblot analysis of ZBP1, CASP1, CASP8, and RIPK3 following immunoprecipitation (IP) with normal immunoglobulin G (IgG) or anti-ZBP1 antibody in cells treated with siRpa1 (n = 3, and representative images are shown). (F) Western blot analysis of pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P43) and activated (P36) caspase-11 (CASP11), pro- (P53) and activated (P34) GSDME, pro- (P35) and cleaved (P17/P19) caspase-3 (CASP3), pro- (P35) and cleaved (P20) caspase-7 (CASP7), pro- (P55) and cleaved (P18) caspase-8 (CASP8), phosphorylated MLKL (pMLKL), total MLKL (MLKL), phosphorylated RIPK3 (pRIPK3), and total RIPK3 (RIPK3) in siRpa1 and siZbp1 treated cells (n = 3, and representative images are shown). (G) TUNEL staining of E12.5 limb buds of WT, CKO, and CKO; Zbp1−/− mice. Scale bars, 500 μm. (H) Quantification of TUNEL-positive cells in (G) (n = 3). One-way ANOVA. (I) Photographs of WT, CKO, and DKO mice at E16.5 (n = 3, and representative images are shown).
Fig 4: Rpa1 knockdown leads to PANoptosis.(A) Representative images of cell death in dimethyl sulfoxide (DMSO), MCC950, Nec-1, and zVAD-FMK, and the combination of MCC950, Nec-1, and zVAD-FMK (N + M + Z)–treated cells. Scale bar, 100 μm. (B) Quantification of cell death in (A) (n = 3). One-way analysis of variance (ANOVA). (C) Western blot analysis of RPA1 and ZBP1 in cells treated with siNC, DMSO, MCC950, Nec-1, zVAD-FMK, and N + M + Z (n = 3, and representative images are shown). (D and E) Western blot analysis of pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P43) and activated (P36) caspase-11 (CASP11), pro- (P53) and activated (P34) GSDME, pro- (P35) and cleaved (P17/P19) caspase-3 (CASP3), pro- (P35) and cleaved (P20) caspase-7 (CASP7), pro- (P55) and cleaved (P18) caspase-8 (CASP8), phosphorylated MLKL (pMLKL), total MLKL (MLKL), phosphorylated RIPK3 (pRIPK3), and total RIPK3 (RIPK3) in siRpa1 treated cells (D) and CKO mice limb bud (E). (D and E, n = 3, and representative images are shown)
Fig 5: Deletion of Rpa1 debilitates limb development.(A) Photographs of Rpa1fl/fl [wild type (WT)] and Prrx1-Cre; Rpa1fl/fl (CKO) mice at postnatal day 0 (P0). (B and C) Representative whole-mount skeletal staining of WT and CKO mice at P0. A, autopod; S, stylopod; Z, zeugopod. Scale bar, 2 mm. (D) Representative images of WT and CKO embryos from E12.5 to E18.5. Scale bars, 2 mm. (E) Limb bud traces of WT and CKO embryos from E12.5 to E18.5. (F) Representative general observation of the WT and CKO mice calvaria. HL, hindlimb; FL, forelimb. (G) Representative skull images from whole-mount skeletal staining preparations of WT and CKO mice at P0. The black dotted circles indicate the missing calvarium (n ≥ 5, and representative results are shown).
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