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. 2020 Nov 12;11(3):892–907.e1. doi: 10.1016/j.jcmgh.2020.11.001

Figure 3.

Figure 3

PAK1 deficiency results in Notch1 activation and increased stemness in colonic crypts of IL10 KO mice. (A) Immunohistochemistry (IHC) showed increased Notch1 pathway activation and expansion of the stem cell compartment, marked by NICD, Hes1, Olfm4, and Lgr5. In addition, β-catenin showed a cytoplasmic accumulation in DKO compared with IL10 KO. Ki67 indicated expansion of the proliferative zone at the crypt base of DKO mice. (B) mRNA analysis from IL10 KO and DKO colons indicated an increase in the Notch1 downstream target Hes1, stem cell markers Lgr5 and Olfm4, and Wnt target β-catenin, while Atoh1 and Klf4 were down-regulated. In addition, mRNA of the proinflammatory cytokine IL6 was highly up-regulated in DKO. (C) Immunoblotting of protein extracts from IL10 KO and DKO colonic samples corroborated IHC findings for NICD, LGR5, and β-catenin. (D) IHC for OLFM4 marking the stem cell zone at the base of the crypt in the small intestine of IL10 and DKO mice. OLFM4-positive colon crypts were found in DKO mice while IL10 KO colonic crypts showed no OLFM4 in any sample. (E) Proliferation assay indicating a higher rate of proliferation in DKO LBOs. Exemplary pictures of LBOs from IL10 KO and DKO mice over the course of 3 days underlining hyperproliferative phenotype. Graph shows means + SD of 4 wells from 2 independent experiments. For statistical data, a 2-tailed Student t test or the Kruskal-Wallis ANOVA was used. ∗P ≤ .05; ∗∗P < .01; ∗∗∗P < .001, ∗∗∗∗P < .0001.