Fig. 3: SENP1 suppresses RIPK1-dependent apoptosis during embryonic fetal liver development.
From: SENP1 prevents steatohepatitis by suppressing RIPK1-driven apoptosis and inflammation
a MEFs were transfected with siRNA pools targeting 14 rate-limiting enzymes/proteins that are involved in SUMOylation process for 48 h followed by treatment with TNFα (T, 1 ng/ml) and (5Z)−7-oxozeaeno (5z7, 100 nM) and cultured for an additional 12 h to induce cell death. Cell death was then measured and Z-score was calculated as described. The screen hits were selected based on the median Z-score of the duplicate plates with cut-offs set at Z-score >2 or <−2. Blue, RIPK1 (positive control). Red, SENP1. Yellow, PIAS1. MEFs (b, c) and primary hepatocytes derived from 2-m-o mice (d, e) were treated with 10 ng/ml TNFα in the presence or absence of Nec-1s (10 μM) for indicated time. Cell death was measured as a function of time by SytoxGreen positivity assay (b, d). The levels of p-S166 RIPK1 and cleaved caspase-3 (CC3) were determined by immunoblotting (c, e). Liver sections from E14.5 embryos of indicated genotype were immunostained for p-S166 RIPK1 (f) and stained for HE (g). TUNEL assay (h) and CC3 IHC (i) were performed on E14.5 liver sections of indicated genotypes. j Quantitative RT-PCR analysis of the mRNA expression of cytokines and chemokines in E14.5 livers of indicated genotypes (n = 6). k Volcano plots showing fold-change and P-value for the comparison of E14.5 Senp1−/− fetal livers versus E14.5 Senp1+/+ fetal livers (n = 3). Differentially expressed genes (fold change >1.5, P value <0.05) are depicted in red (upregulated) and blue (downregulated). l Quantitative RT-PCR analysis of the mRNA expression of Ripk1 (n = 6). Heatmap (m) and gene ontology (GO) analysis (n) of 278 genes that are upregulated in E14.5 Senp1−/− fetal livers (vs Senp1+/+ fetal livers) and downregulated in Senp1−/−;Ripk1D138N/D138N fetal livers (vs Senp1−/− fetal livers) (n = 3). n = 8 embryos (f–i), n = 3 biologically independent experiments (b–e). Data are represented as mean ± s.d. (b, d), and mean ± s.e.m. (h, i, j, l). One-way ANOVA, post hoc Dunnett’s test (h, i, j, l). Source data are provided in Source Data file.
