Fig. 1: Sod1 loss suppresses organoid proliferation and survival.
From: Non-enzymatic role of SOD1 in intestinal stem cell growth
A Experimental scheme for generation of Sod1f/f; Vil-creERT2 mice. B Genotyping using PCR. C Western blot analysis of SOD1 protein in organoids from day 0 to day 5 after tamoxifen treatment. Sod1f/f and Sod1f/f; Vil-creERT2 organoids were treated with 2 μg ml−1 tamoxifen on day 0 (2 days after seeding) and analyzed for SOD1 expression from day 0 to day 5 (n = 4 mice per genotype). D Quantification of the percentage of dead organoids from day 0 to day 5 following tamoxifen treatment. TAM, tamoxifen. Data represent mean ± standard error of the mean (SEM), n = 6 wells per mouse; n = 4 mice per genotype. E Western blot analysis of SOD1 protein in organoids after 5 days of tamoxifen treatment (n = 4 mice per genotype). F Confocal images (nuclear staining, blue; and EdU staining, red) of Edu incorporation (1 h) in organoids after 5 days of tamoxifen or ethanol-only vehicle control treatment. Tamoxifen was dissolved in ethanol. EtOH, ethanol. G Brightfield images of organoids after 5 days of tamoxifen or ethanol-only vehicle controls treatment. Green arrowhead indicates “poor” organoids, and red arrowhead indicates dead organoids. H Percentage of organoids with 0, 1, 2, 3, or ≥4 crypts formed after 5 days of tamoxifen treatment. Data represent mean ± SEM; n = 6 wells per mouse; n = 4 mice per genotype; number of organoids counted: n = 504 (Sod1f/f), n = 557 (Sod1f/f;Vil-creERT2) organoids per group; one of three experiments. I Percentage of dead, poor, and healthy organoids analyzed after 5 days of tamoxifen or ethanol-only vehicle controls treatment. Data represent mean ± SEM; n = 6 wells per mouse; n = 4 mice per genotype; number of organoids counted: n = 511 (Sod1f/f + ethanol), n = 656 (Sod1f/f;Vil-creERT2 + ethanol), n = 498 (Sod1f/f + tamoxifen), n = 784 (Sod1f/f;Vil-creERT2 + tamoxifen) organoids per group; one of three experiments. J Fluorescence immunostaining to detect cleaved caspase-3 (yellow), cleaved caspase-8 (green) and cleaved caspase-9 (red) in the organoids after 5 days of tamoxifen treatment. Representative images of staining performed on organoids derived from 3 separate mice. K qPCR results showing relative mRNA expression of Sod1 and Lgr5 genes in organoids 3 and 5 days after tamoxifen treatment. Data represent mean ± SEM. n = 4 mice per genotype. L Organoids cultured for 2 days with Sod1-deficient crypts isolated from the Sod1f/f and Sod1f/f; Vil-creERT2 mice after 4 weeks post 5 consecutive tamoxifen injection (left); histogram showing quantification of the number of dead organoids on the indicated day after seeding (right). Data represent mean ± SEM. n = 3 mice per genotype. All results are representative of at least three independent experiments. All statistical significances were tested by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.
