Extended Data Fig. 4: LXR activation upregulates EGFR ligands in IECs.

(a) qPCR analysis of Abca1 expression in organoids treated with either DMSO or GW3965 for 5 days in ENR culture medium. (b) Left: Scheme showing sorting of GFP⁺ intestinal stem cells (ISCs) from Lgr5-eGFP-creERT2 mice and Paneth cells (PCs) from WT (non-Lgr5) mice for organoid co-culture. Right: representative pictures and quantification of average number of buds/organoid and % of organoids with the indicated number of buds from ISC-PC organoid co-culture treated with either DMSO or GW3965. The diagram was adapted from ref. ⁷². (c) qPCR analysis of niche signals in SI organoids from WT mice treated with either DMSO or GW3965 in ENR culture medium. (d-e) qPCR analysis of niche factors (d) or EGFR ligands (e) in SI crypts isolated on day0, day1 and day3 post-irradiation from WT mice fed with either STD or GW3965 diet. Datasets for 1 and 3dpi are normalized to their corresponding 0dpi. (f) Representative pictures and quantification of average number of buds/organoid and percentage of organoids with the indicated number of buds in SI organoids cultured in ENR or NR (Noggin and R-spondin only) medium and treated with DMSO or GW3965. (g) Scheme of the experiment shown in Main Fig. 2d-f. Briefly, SI crypts were isolated from WT mice and cultured in ENR or NR media +/− GW3965 for 5–7 days (primary organoids). Organoids were then digested to single cell suspension and 10,000 live cells were re-seeded for secondary organoid culture. Secondary organoids were cultured in ENR or NR medium and stimulated with DMSO or GW3965 according to their treatment protocol in primary cultures. Secondary cultures were imaged longitudinally using Incucyte live imaging and plating efficiency, average number of buds/organoid and % of budding organoids was assessed at day 4–7 of culture. (h) Representative immunoblot of pERK1/2, tERK1/2 and vinculin from organoids cultured in ENR or NR and treated with either DMSO or GW3965. Western blot quantification of the ratio between phospho-ERK (pERK) and ERK (normalized to Vinculin) from organoids cultured for 5 days with DMSO or GW3965 in ENR or NR media. Vinculin was used as a loading control in the same gel as phospho- and total- ERK1/2 and was cut out to probe with anti-vinculin antibody due to molecular weight difference with tERK1/2 or pERK1/2. For gel source data, see Supplementary Fig. 3. Data are representative of three (b, d, e and h), four (a, c) independent experiments with 3–8 mice/group (each dot represents one mouse). Data are shown as mean ± s.e.m. (a-f, h). Significance was assessed by unpaired two-sided t test in a, b (buds/organoid) and c-e), paired two-sided t test (h), two-way ANOVA with Bonferroni test (b, de novo buds) and one-way ANOVA with Tukey’s post hoc test (f). Part of the schematic in panel g was drawn using BioRender.com.

Source Data