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. Author manuscript; available in PMC: 2020 May 1.
Published in final edited form as: Nat Cell Biol. 2019 Nov 4;21(11):1321–1333. doi: 10.1038/s41556-019-0402-6

Fig. 3. TET1 catalytic activity is required for liver organoid initiation and maintenance.

Fig. 3

a, FACS-sorted EpCAM+ ductal cells freshly isolated from WT undamaged livers were transfected with a pool of siRNAs, each of them targeting specifically a selected epigenetic modifier, and organoid formation efficiency was evaluated 10 days later. Results are shown as percentage of organoid formation efficiency compared to mock transfected cells. The graph represents mean±SD of n=3 independent experiments (dots). p-values were calculated using one-way ANOVA in conjunction with Tukey’s HSD test by comparison to siCtrl. *, p= 0.01031057 siTet1 vs siCtrl,. b, FACS-sorted EpCAM+ ductal cells derived from RosaCreERT2 x Tet1flx/flx mouse livers were plated in organoid isolation medium supplemented with 5μM hydroxytamoxifen or vehicle and organoid formation efficiency was evaluated 6 days later. Representative bright field images are shown. Data are reported as percentage of organoid formation compared to Cre-Tam- cells. Graphs represent mean±SD of n=3 independent experiments. p-value was calculated using Student's two-tailed t-test vs Cre-Tam- (*, Cre-Tam+ p=0.03781815; ***, Cre+Tam+ p=4.812E-05). c-e, EpCAM+ ductal cells isolated from Tet1 hypomorphic mice were used to generate liver organoids (Tet1hypo/hypo, blue) or were transfected with a hTET1 full length cDNA (hypo-OE organoids, red) or catalytically inactive hTET1 H1671Y/D1673A (hypo-OEcat.mut. organoids, turquoise). Organoids derived from WT littermates were used as controls (black). c, Scheme indicates the lines generated. d, Western blot analysis of TET1 protein levels. The graph represents TET1 levels. Complete blot is shown in data source. Results are presented as mean±SD of n=3 independent experiments (dot). **, p-value calculated using Student's two-tailed t-test vs WT (Tet1hypo/hypo p=0.006779543). e, Representative bright field images of WT (n=2), Tet1hypo/hypo(n=4) hypo-OE (n=1) and hypo-OEcat.mut. (n=1) organoid lines at passage 3. Graph indicates passage number.