Extended Data Fig. 5: Characterizing the in vitro response of intestinal organoids to TGFβ pathway stimulation.
a) Representative immunofluorescent images of Clu-GFP organoids treated with TGFβ1 or SB431542 (10 µM) for 24 hours, as indicated. Staining shows EpCam, Clu-GFP, and Smad2/3. Nuclear Smad2/3 is indicated by solid arrows, nuclear holes indicated by open arrows. Scale bar,10 µm. N = 3 replicates. b) Gating strategy identifying GFP+ cells isolated from organoids via flow cytometry. Plots indicate cell populations that were included based on size (singlets) and viability (DAPI-) (left). Representative plots indicating the GFP+ fraction in (1) GFP negative organoids or (2) Clu-GFP organoids treated with SB431542 (10 µM) or induced with TGFβ1(200 pM) (left). SS, side scatter; FS, forward scatter. c) Organoids expressing Clu-GFP were treated with TGFβ1 (200 pM; 24 or 48 h) and the percent GFP+ cells quantified by flow cytometry. Bars represent the mean ( ± SEM) of N = 3 individual experiments performed in triplicate, N = 5 for control and 48 h treatment groups (dots). Unpaired, two-tailed t-test; SB P = 0.25, 24 h TGFβ1 P = 0.04, 48 h TGFβ1 P = 0.01. d) Representative IF images of Lgr5-GFP-expressing organoids treated +/- TGFβ1 (50 or 200pM; 48 h, as indicated). Scale bar, 50 µm (left, middle), Scale bar, 10 µm (right). e) Quantification of the number of organoids in culture following 24 h exposure. Organoids were treated for 24 h after 3 days of growth and the total number of organoids/well quantified. N = 3 independent experiments performed in triplicate (dots). All data shown as mean ± SEM. One-way ANOVA, P = 0.54. Source numerical data available in source data.
