Extended Data Fig. 3: TET1 catalytic activity is required for liver organoid formation and maintenance.

a, Tet1 and Lgr5 mRNA levels (n = 3 mice). Student’s two-tailed t-test statistical analyses were performed versus undamaged. b, Tet1 mRNA levels (24 h after transfection) and organoid formation efficiency 10 days after Tet1 siRNA knock-down using 4 independent Tet1 siRNAs. Data is presented as percentage relative to siCtrl. Graph indicates mean ± SD of n = 3 independent experiments. Student’s two-tailed t-test statistical analyses were performed versus mock-transfected cells. c, Scheme of the two different Tet1 alleles used. d, Tet1 mRNA levels in WT, Tet1^hy/+ and Tet1^hy/hy and Tet1 conditional knock-out (cKO) organoids presented as mean ± SD of n = 3 experiments. e, Representative Western blot image showing TET1 protein levels in WT, Tet1^hy/+ and Tet1^hy/hy organoids (3 independent experiments). f, Organoid formation efficiency from FACS-sorted EpCAM⁺ cells derived from RosaCre^ERT2 x Tet1 ^flx/flx livers treated with 5 μM hydroxytamoxifen (mean ± SD of n = 3 independent experiments). Student’s two-tailed t-test statistical analyses were performed versus non-induced control. g, Whole mount immunofluorescence staining of 5hmC (green) on WT, Tet1^hy/hy, hypo-OE and hypo-OE^cat.mut. organoids. Representative images are shown (2 experiments). Scale bar, 50 μm. h, Graph represents organoid size at the indicated passages (mean ± SD of n = 3 independent experiments). Student’s two tailed t-test statistical analyses were performed versus WT. i, Growth curves. j, Organoid formation efficiency at the indicated passage expressed as a percentage of organoids. Graphs represent mean ± SD of n = 3 independent experiments. Student’s two tailed t-test statistical analyses were performed versus WT. k, Representative confocal images of Cleaved Caspase 3 whole mount immunostaining on WT, Tet1^hy/hy, hypo-OE and hypo-OE^cat.mut. organoids (2 independent experiments). Scale bar, 25 μm.

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