The non-muscle-myosin-ii heavy chain Myh9 mediates colitis-induced epithelium injury by restricting Lgr5+ stem cells

Transcription

1 Supplementary Information The non-muscle-myosin-ii heavy chain Myh9 mediates colitis-induced epithelium injury by restricting Lgr5+ stem cells Bing Zhao 1,3, Zhen Qi 1,3, Yehua Li 1,3, Chongkai Wang 2, Wei Fu 2 and Ye-Guang Chen 1,* 1 The State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing , China; 2 Department of General Surgery, Peking University Third Hospital, Beijing , China 3 These authors contributed equally to this work. * Corresponding author: Ye-Guang Chen, PhD School of Life Sciences Tsinghua University Beijing China Tel.: Fax: ygchen@tsinghua.edu.cn Supplementary Figures

2 Supplementary Figure 1 Supplementary Figure 1 Examination of genotypes and Myh9 mrna levels in gut epithelium confirmed the knockout of one Myh9 allele in Myh9 fl/+ ;Villin-cre mice. (a) The gut epithelia of 8-week-old Myh9 +/+ ;Villin-cre and Myh9 fl/+ ;Villin-cre mice were harvested for genotyping using indicated primers. (b) The gut epithelia of Myh9 +/+ ;Villin-cre and Myh9 fl/+ ;Villin-cre mice were harvested to examine Myh9 expression using quantitative RT-PCR. Gapdh was used as an internal control. The data represent the mean ± S.D. (n=3). *** indicates p<

3 Supplementary Figure 2 Supplementary Figure 2 Confocal cross-sectioning shows the intestinal and colonic epithelium of Myh9 +/+ ;Villin-cre and Myh9 fl/+ ;Villin-cre mice. The intestine (a) and distal colon (b) were visualized by immunofluorescence with anti-myh9 antibody (green) and anti-ephb2 antibody (red). Bottom: enlargements of the square areas. Nuclei were counter-stained with DAPI (blue). Scale bar, 100µm. 3

4 Supplementary Figure 3 Supplementary Figure 3 Proliferation status and cell apoptosis in DSS-treated Myh9 +/+ ;Villin-cre and Myh9 fl/+ ;Villin-cre mice colonic epithelium. Myh9 +/+ ;Villin-cre and Myh9 fl/+ ;Villin-cre mice were treated with 3% DSS for 5 days followed by 2-day recovery before sacrificed. The distal colon tissues were subjected to Ki67 staining (a) and Tunel assay (b) to detect proliferation status and cell apoptosis in distal colonic epithelium. Bottom: enlargements of the square areas. Scale bar, 100µm. 4

5 Supplementary Figure 4 Supplementary Figure 4 Knockdown of Myh9 expression using adenovirus-based shrna promoted the survival of isolated crypts. (a) The isolated mouse intestinal crypts infected with adenovirus expressing nonspecific (NS) or Myh9 shrna were cultured in the presence of ENR for 36 hours and harvested to examine Myh9 expression using quantitative RT-PCR. Gapdh was used as an internal control. (b) One hundred intestinal crypts isolated from 8-week-old mice were infected with adenovirus expressing NS or Myh9 shrna and cultured in the presence of ENR for 36 hours. Statistical analysis of numbers of survived crypts was shown. (c) Blebbistatin promotes the survival of isolated human colonic crypts. One hundred human colonic crypts isolated from normal human colon tissues were embedded in 50µl Matrigel and cultured in the presence of ENR for 36 hours. DMSO (mock) or blebbistatin (10µM) was added in crypts culture medium 5

6 immediately after polymerization of Matrigel. Statistical analysis of numbers of survived crypts was shown. The data represent the mean ± S.D. (n=3). *** indicates p< Scale bar, 50 µm. 6

7 Supplementary Figure 5 Supplementary Figure 5 Blebbistatin accelerates the proliferation of Lgr5+ stem cells in organoid cultures. Fifty Lgr5-EGFP-IRES-creERT2 organoids were cultured for 4 days before treated with DMSO (mock) or blebbistatin (10µM) for 24 hours. Then, the single cells were dissociated and subjected to FACS to calculate the number of Lgr5-GFP+ stem cells per organoid. The data represent the mean ± S.D. (n=3). ** indicates p<

8 Supplementary Figure 6 Supplementary Figure 6 Blebbistatin activates Akt in Lgr5+ intestinal stem cells. Single Lgr5-GFPhi cells embedded in Matrigel were treated with the crypt culture medium containing ENR, Wnt3a and DMSO (mock) or 10µM blebbistatin for 12 hours and subjected to anti-phospho-akt antibody staining followed by FACS analysis. 8

9 Supplementary Figure 7 Supplementary Figure 7 Akt activity is essential for Myh9 deficiency to promote crypts survival. Intestinal crypts isolated from Myh9 +/+ ;Villin-cre and Myh9 fl/+ ;Villin-cre mice were cultured in the ENR medium together with DMSO (mock) or 10µM blebbistatin for 24 hours and counted. Statistical analysis of numbers of survived crypts was shown. The data represent the mean ± S.D. (n=3). *** indicates p<

10 Supplementary Figure 8 Supplementary Figure 8 Blocking actin-myosin contraction has no effect on crypt survival. (a) NIH3T3 cells treated with DMSO (mock), 10µM blebbistatin, 100nM cytochalasin D or 10nM swinholide A for 4 hours were fixed for actin filaments staining with rhodmine phalloidin. (b) The isolated crypts were cultured in the presence of ENR, together with 10µM blebbistatin, 100nM cytochalasin D or 10nM swinholide A for 24 hours. (c) The isolated crypts were cultured in the presence of ENR, together with 10µM blebbistatin or 10µM ML-7 for 24 hours. The data represent the mean ± S.D. (n=3). *** indicates p<

11 Supplementary Figure 9 Supplementary Figure 9 Withdrawal of Noggin increases Id1 mrna expression in passaged Lgr5 organoids. Intestinal organoids cultured in the ENR medium were dissociated from Matrigel and passaged in the medium with ENR or ER for the indicated time. Then, the organoids were harvested to examine Id1 expression using quantitative RT-PCR. Gapdh was used as an internal control. The data represent the mean ± S.D. (n=3). *** indicates p<

12 Supplementary Figure 10 Supplementary Figure 10 Blebbistatin defended the Lgr5+ colonic stem cells independently of any anti-inflammatory effect. 8-week-old Lgr5-EGFP-IRES-creERT2 mice fed with water or 1.5% DSS for 5 days followed by 2-day recovery were daily injected with mock or blebbistatin, and Lgr5+ colonic stem cells and CD45+ immune cells in distal colon were examined by confocal cross-sectioning. Scale bar, 100 µm. 12

13 Supplementary Figure 11 Supplementary Figure 11 Full immunoblots. 13