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. 2019 Mar 6;10:1071. doi: 10.1038/s41467-019-09060-w

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Znhit1 is essential for Lgr5+ ISC maintenance thus intestinal homeostasis. a Eight-week-old Villin-creERT and Znhit1^fl/fl; Villin-creERT mice were daily injected with tamoxifen for 4 days followed by 7-day waiting period. Top: Scheme of Cre induction strategy. Bottom: Body weight comparison between Villin-creERT and Znhit1^fl/fl; Villin-creERT mice at indicated time following tamoxifen treatment. (n = 5 mice per genotype). b Kaplan–Meier survival curves of Villin-creERT and Znhit1^fl/fl; Villin-creERT mice post tamoxifen administration (n = 9 mice per genotype). c Olfm4 in situ was performed in intestinal sections from Villin-creERT and Znhit1^fl/fl; Villin-creERT mice following tamoxifen treatment. d Intestinal crypts were isolated from Villin-creERT and Znhit1^fl/fl; Villin-creERT mice following tamoxifen treatment, embedded in Matrigel (100 crypts per well) and cultured for 2 days. The statistical analysis of organoid numbers (n = 3 mice per genotype) was shown. e Eight-week-old Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 mice were daily injected with tamoxifen for 3 days followed by 4-day waiting period, then Lgr5+ ISCs were examined by confocal cross-sectioning. The GFP+ cells were quantified for statistical analysis (n = 3 mice per genotype). f Intestinal crypts were isolated from Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 (+/+) and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 (fl/fl) mice following tamoxifen treatment for immunoblotting with the indicated antibodies. GAPDH served as a loading control. The statistical data represent mean+ s.d. (n = 3 mice per genotype). g Intestinal crypts were isolated from Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 (+/+) and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 (fl/fl) mice following tamoxifen treatment, embedded in Matrigel (100 crypts per well) and cultured for 7 days. The organoid buddings were quantified for statistical analysis (n = 5 mice per genotype). h Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 mice were daily injected with tamoxifen for 4 days followed by 7-day waiting period. Body weight comparison was shown (n = 3 mice per genotype). The statistical data represent mean ± s.d. Student’s t-test: ***P < 0.001. All images are representative of n = 3 mice per genotype. Scale bar, 50 μm