Extended Data Fig. 3: Effects of Cysteine-rich diet on ISC and progenitor cells.
From: Dietary cysteine enhances intestinal stemness via CD8+ T cell-derived IL-22
a, IHC detection for intestinal stem cell and progenitor cell proliferation (4-h BrdU pulse), crypts regeneration (OLFM4), and post-mitotic Paneth cell identity (Lysozyme, LYZ) from control- and CysRD-fed mice in homeostasis. Scale bar, 20 µm. b, Quantification of BrdU+ cells per crypt (n = 40), OLFM4+ crypt length (n = 14), and LYZ+ Paneth cells per crypt (n = 40) from 5 control and 5 CysRD diets treated mice under homeostasis condition. c, Villi length and crypt depth in the small intestine isolated from control- and CysRD-fed mice in homeostasis (n = 14 per group). d, Flow cytometry for Lgr5-GFP gating panel and quantification of ISCs (Lgr5-GFPhi) and progenitors (Lgr5-GFPlow) frequency from 6 weeks control- and CysRD-fed mice. n = 6 for each group. e, Representative crypt images of Hmgcs2, Lgr5 mRNAs (ISH) and proteins (IF) from control- and CysRD-fed mice intestine. Scale bar, 10 µm. f, Schematic (top) of the intestinal renewal assay including the timeline of dietary regimens, tamoxifen induction, and tissue collection in the Lgr5-IRES-CreERT2; Rosa26LSL-tdTomato lineage tracing reporter mice. Representative images (bottom) of IHC for tdTomato+ Lgr5+ ISC-derived progenies in the small intestine. Quantification (right) of day-3 RFP+ length (n = 100, 20 data points per mouse) from 5 control and 5 CysRD diets treated mice. Scale bar, 50 µm. g, Schematic (left) of the intestinal permeability assay including the timeline of dietary regimens, irradiation (XRT, 7.5 Gy x 2), and tissue collection in the WT/B6 mice. Quantification (right) of plasma FITC-dextran levels 4 h after oral gavage in unirradiated and irradiated cohorts (n = 5 per group). h-i, Immunoblots (h) and IHC (i) analyses of Cleaved Caspase-3 (CC3-Asp175) in small intestine crypts from control- and CysRD-fed mice, assessed both at baseline and 8 h after irradiation. Scale bar, 20 µm. Unpaired two-tailed Student’s t-tests (b, c, d, f, g, i). Data (b, c, d, f) are mean ± s.d. Box and whiskers (g, i) are plotted min to the max value.
