Extended Data Fig. 8: Effect of cholesterol and LXR signalling in organoids from multiple tissue types.

(a) Representative pictures, quantification of average number of buds/organoid and quantification of % of organoids with indicated number of buds in SI organoids treated with vehicle or cholesterol from Villin-Cre:LXRα^f/fβ^f/f (LXR^ΔIEC) mice and their littermate LXRαβ^flox/flox controls. (b) WT SI organoids were cultured for 5 days in NR medium with either DMSO or RGX-104. Left: Representative pictures and quantification of average number of buds/organoid. Right: qPCR analysis of Abca1 expression. (c) qPCR analysis of the expression of EGFR ligands in organoids treated with either DMSO or RGX-104. (d) Scheme of the experiment shown in (e-g): WT colon primary organoids were cultured for 5-6 days with either DMSO or GW3965 (+exogenous Wnt for the first three days). Organoids were then split and 10,000 live colonocytes were re-seeded for secondary organoids, treated with DMSO or GW3965 (as in the primary cultures) for 6-7 days. Organoids were imaged longitudinally with Incucyte live imaging and plating efficiency, and organoid area was measured at the end of the experiment. (e-g) Representative pictures (e), quantification of plating efficiency (f) and total organoid area over time (g) of WT organoids as described in d. For organoid area analyses over time, same wells were imaged every 4 h for a peiod of 6-7 days. (h) qPCR analysis expression of Abca1 and Areg in colonic organoids treated with DMSO or GW3965. (i) WT salivary gland organoids were cultured for 4-6 days with either DMSO or GW3965. Representative pictures, quantification of the organoids area/well and qPCR analysis of Abca1 and Areg. (j) Scheme showing WT mice fed with either standard (STD) or GW3965 diet for 10 days followed by focal irradiation of the neck region targeting salivary glands. Representative H&E and zoomed inset of salivary glands from mice fed with STD or GW3965 diet at 0 dpi (left, 10 days after the diet) and at 14 dpi (right, 14 days after the irradiation). Measurement of salivary gland duct area was used as a proxy for salivary gland regeneration. Data are representative of 2–4 independent experiments and each dot represents one mouse, except for f where each dot represents one well. Data are shown as mean ± s.e.m. (a-i). In j, data are shown as mean ± s.e.m. for the bar plot and median ± quartile for the violin plot. Significance was assessed by unpaired two-sided t test (b, c, f, h and i), unpaired two-sided t-test between the slopes for DMSO and GW3965 treated organoids obtained by linear regression analysis (g), and one-way ANOVA with fisher’s LSD post hoc test (a). Part of the schematic in panel d was drawn using BioRender.com and part of the schematic in panel j was adapted from ref. ¹⁹, CC-BY 4.0.

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