Figure 4. MTGR1 loss increases enteroid apoptosis, but effects cannot be rescued by apoptosis inhibition.

(A) Schematic of RNA-sequencing experiment of crypts and enteroids. n = 3 mice per genotype. (B) GSEA of “Hallmark” collection apoptosis-related genes in Mtgr1^−/− enteroids at day 1 (left) and day 3 (right) post-plating. NES = normalized enrichment score. (C) Enteroids were fixed and embedded at day 1 post-plating, and apoptotic cells were marked by immunofluorescent staining against cleaved caspase-3 (CC3, red). β-catenin (green) and DAPI (blue) were used for co-staining. Quantification shown as percent CC3-positive cells per high powered field (HPF). Scale bar = 200μm. n = 8–9 HPFs per genotype. (D) Enteroids were plated and overlaid with media containing indicated concentrations of the caspase inhibitor, Z-VAD-FMK, or (E) the necrosis inhibitor, necrostatin. Enteroids were counted daily and normalized to day 1 numbers. n ≥ 6 wells per condition. (F) GSEA of “Hallmark” collection p53 pathway-related genes in Mtgr1^−/− enteroids at day 1 (left) and day 3 (right) post-plating. (G) Enteroids were plated and overlaid with media containing the p53 inhibitor, pifithrin, as indicated. n ≥ 3 wells per condition. *P<0.05, ****P<0.0001, Student’s t test (C) or two-way ANOVA (D, E, G), significance indicated by FDR q value (B, F).