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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. 3 — Znhit1 controls the transcription of Lgr5+ ISC fate-determining genes. a, b Eight-week-old Villin-creERT (+/+) and Znhit1^fl/fl; Villin-creERT (fl/fl) mice were daily injected with tamoxifen for 4 days followed by 7-day waiting period. Intestinal crypts were harvested for RNA-seq (a) and qRT-PCR (b) to analyze the gene expression changes. Clustered heatmap of log2-transformed RPKMs shows the differentially expressed genes after Znhit1 deletion. Log2-transformed fold changes of indicated genes were marked in right. c Tgfb1 and Tgfbr2 staining of intestinal sections from Villin-creERT (+/+) and Znhit1^fl/fl; Villin-creERT (fl/fl) mice following tamoxifen treatment. d Eight-week-old Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 (+/+) and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 (fl/fl) mice were daily injected with tamoxifen for 3 days followed by 4-day waiting period. Intestinal crypts were harvested to examine the expression of indicated genes using qRT-PCR. e Phospho-Smad2 staining of intestinal sections from Villin-creERT (+/+) and Znhit1^fl/fl; Villin-creERT (fl/fl) mice following tamoxifen treatment. f Intestinal crypts were isolated from Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 (+/+) and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 (fl/fl) mice following tamoxifen treatment and subjected to in vitro culture in the presence of 3 μM CHIR99021 and/or 10 μM SB431542 for 4 days. Mock: DMSO. g The cultured organoids were harvested to examine the expression of indicated genes using qRT-PCR. For qRT-PCR, histone H3 was used as an internal control. The statistical data represent mean ± s.d. (n = 3 mice per genotype or treatment). Student’s t-test: ***P < 0.001. **P < 0.01. All images are representative of n = 3 mice per genotype. Scale bar, 50 μm