Fig. 4: SOD1 supports organoid growth by suppressing WNT signaling and excessive Paneth cell differentiation.
From: Non-enzymatic role of SOD1 in intestinal stem cell growth
A Relative expression of Paneth cell signature genes by RNA-seq analysis are shown as a histogram comparing Sod1ΔIEC and control organoids after 3 days of tamoxifen induction. Sod1f/f and Sod1f/f; Vil-creERT2 organoids were harvested after 3 days of tamoxifen induction for RNA-seq. n = 3 mice per genotype. B Fluorescence immunostaining to detect LYZ expression (red) in the organoids after 3 days of tamoxifen induction. Arrowheads indicate location of LYZ+ Paneth cells. Images are representative of stainings performed on organoids derived from 3 separate mice (left panel); Histogram (middle panel) shows the quantification of the number of LYZ+ Paneth cells per crypt, according to the scheme in the inset (right panel). The number of crypts measured per group was labeled on the histogram. One of three experiments. Data represent mean ± SEM; n = 6 wells per group; n = 3 mice per genotyping. Student t-test (two-tailed). ***P < 0.001. C qPCR analysis of Sod1, Lyz, and Wnt3a genes after 3 days of tamoxifen induction. Data represent mean±SEM; n = 3 mice per genotyping. Student t-test (two-tailed). **P < 0.01, ***P < 0.001. D Representative images of Sod1f/f and Sod1f/f; Vil-creERT2 organoids treated with tamoxifen and cultured in reducing RSPO1 concentrations for 5 days. E Percentage of dead organoids assayed after 5 days in culture using indicated concentrations of RSPO1. Data represent mean±SEM; n = 6 wells per group; n = 3 mice per genotyping. Statistical significances were tested by two-way ANOVA. ***P < 0.001. Organoids number counted: n = 246 (Sod1f/f + normal RSPO1), n = 385 (Sod1f/f; Vil-creERT2 + normal RSPO1), n = 244 (Sod1f/f + 1/2 RSPO1), n = 328 (Sod1f/f; Vil-creERT2 + 1/2 RSPO1), n = 256 (Sod1f/f + 1/4 RSPO1), n = 303 (Sod1f/f; Vil-creERT2 + 1/4 RSPO1), n = 265 (Sod1f/f + 1/8 RSPO1), n = 281 (Sod1f/f; Vil-creERT2 + 1/8 RSPO1), n = 229 (Sod1f/f + 1/16 RSPO1), n = 275 (Sod1f/f; Vil-creERT2 + 1/16 RSPO1) organoids per group; one of three experiments. F Quantification of percentage of organoids with 0, 1, 2, 3, or ≥4 crypts formed after 5 days in culture using indicated concentrations of RSPO1. Data represent mean±SEM; n = 6 wells per group; n = 3 mice per genotyping; organoids number counted: n = 189 (Sod1f/f + normal RSPO1), n = 203 (Sod1f/f; Vil-creERT2 + normal RSPO1), n = 197 (Sod1f/f + 1/2 RSPO1), n = 185 (Sod1f/f; Vil-creERT2 + 1/2 RSPO1), n = 186 (Sod1f/f + 1/4 RSPO1), n = 181 (Sod1f/f; Vil-creERT2 + 1/4 RSPO1), n = 196 (Sod1f/f + 1/8 RSPO1), n = 203 (Sod1f/f; Vil-creERT2 + 1/8 RSPO1), n = 166 (Sod1f/f + 1/16 RSPO1), n = 186 (Sod1f/f; Vil-creERT2 + 1/16 RSPO1) organoids per group; one of three experiments. All results are representative of at least three independent experiments.
