Supplementary Figure 6: Intestinal adenoma development by CRAD KO. | Nature Cell Biology

Supplementary Figure 6: Intestinal adenoma development by CRAD KO.

From: Deregulation of CRAD-controlled cytoskeleton initiates mucinous colorectal cancer via β-catenin

Supplementary Figure 6

(a), CRISPR/Cas9-mediated targeting of CRAD alleles. Exon2 of CRAD was targeted using gRNAs. gRNA and Cas9 mRNA were injected into the pronuclei of C57BL/B6 mouse embryos. (b), CRAD expression in the small intestine. IHC of mouse intestine. (c), Validation of CRAD KO. The cell lysates were extracted from the small intestine of six independent CRAD KO mice for IB. (d), Sequencing analysis of potential off-target genes of CRAD gRNAs. No mutations in 14 off-target genes were detected. (e), Increased Wnt signaling target genes in CRAD KO mouse. CRAD KO-induced tumors were analyzed for qRT-PCR of Hippo, Notch, Shh, BMP, and Wnt signaling pathway target genes. n = 3 independent mice. f,g, Tumor development in the lung and pancreas of CRAD KO mice. Development of the early lesion of SCLC. The multiple sites displaying the early lesion of SCLC (black arrows; f) and the pancreatic tumors (g; i and ii) were observed. Scale bars indicate 100 μm (f) or 20 μm (g). (h), Disruption of epithelial cell integrity. Cytokeratin 19 (CK19). Arrows: Villi not expressing CK19. (i and j), Analysis of inflammation in CRAD KO mouse. Small intestine samples of WT and CRAD KO mouse were stained with H&E (i; n = 8 mice). Intestinal inflammation (foci size > 200 μm) was assessed by counting (j). Scale bars indicate 200 μm. (k-n), Cell hyperproliferation in CRAD KO small intestine. Phospho-histone H3 (k) and Ki67 (n) staining and quantification (l and m; n = 10 crypts). Scale bars indicate 20 μm. o, Analysis of apoptosis in CRAD KO-induced tumor lesion. WT and CRAD KO mice were analyzed for cleaved Caspase-3 (Casp3). Scale bars indicate 20 μm. (p), Abnormal differentiation of IECs by CRAD KO. WT and CRAD KO small intestine were immunostained with ChgA (arrows). Scale bars indicate 20 μm. (q), Disorganized cell adhesion in CRAD KO mice. Cells were stained with E-cadherin. Scale bars indicate 20 μm. (r), Increased β-catenin target genes in the intestinal adenoma of CRAD KO mice. IHC for CD44. Scale bars indicate 20 μm. Representative images of three independent experiments; Error bars: mean +/− S.D.; NS: not significant (P > 0.05); Two-sided unpaired t-test.

Back to article page