Journal: bioRxiv
Article Title: Chromatin Remodelling in Damaged Intestinal Crypts Orchestrates Redundant TGFβ and Hippo Signalling to Drive Regeneration
doi: 10.1101/2024.08.30.610472
Figure Lengend Snippet: a) Representative images of Clu-tdTom;Lgr5-GFP organoids treated with TGFβ1 (200 pM) and 4-OHT (schematic), with Paneth, Enteroendocrine and Goblet cells identified by staining for Lyz1, ChgA, and Muc2 (red), respectively. Scale bars, 10 µm (Crypt view) and 50 µm (complete organoid). Red arrows indicate cells of interest. b) Schematic (top) and representative IF images (bottom) of control and 5-FU-treated (20 µM; 24 h) Lgr5-GFP expressing organoids. Scale bar, 10 µm (Crypts), 50 µm (whole organoids). c) Schematic (top) and brightfield images (bottom) showing organoid morphology following a 24-hour pulse of 5-FU (20 µM) and after 2 successive passages. Scale bar, 10 µm. d) Expression of ISC signature genes in organoids treated with 5-FU (20 µM; 48 h) quantified by RT-qPCR and normalized to Ywhaz expression. Bars indicate mean ± S.E.M of three independent experiments performed in triplicate. Unpaired, two-tailed t-test; ***p<0.001 e) Expression of revSC signature genes in organoids treated with 5-FU (20 µM; 48 h), quantified by RT-qPCR, and normalized to Ywhaz expression. Bars indicate mean ± S.E.M of three independent experiments performed in triplicate. Unpaired, two-tailed t-test; ***p<0.001 f) Clu-CreERT2;lsl-tdTom;Lgr5-GFP organoids treated with 5-FU (20 µM) and 4-OHT (top) as indicated (Schematic), and lineage-traced offspring visualized after damage, as indicated. Scale bars, 10 µm.
Article Snippet: The following primary antibodies were used: rabbit anti-lysozyme (Dako, #2230, 1:1,000), rabbit anti-Muc2 (Santa Cruz, #sc-15334, 1:300), rabbit anti-Olfm4 (Cell Signaling, #66479, 1:300), rabbit anti-ChgA (Abcam, #ab85554, 1:250), goat anti-GFP (Abcam, #ab5450, 1:1000), chicken anti-GFP (Abcam, #ab13970; 1:1000) Rabbit anti-ki67(Abcam, #ab15580), Anti-CD326 (Biolegend, #118205), Rabbit anti-Smad2/3 (Cell Signaling, #8685S, 1:300), Rabbit anti-pSmad2 (Cell signaling, #18338, 1:200), Rabbit anti-Smad7 (Abcam, #ab216428; 1:100).
Techniques: Staining, Control, Expressing, Quantitative RT-PCR, Two Tailed Test
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