Fig 2: Notch1 is a direct target of miR-744 in CRC. (A) Predicted and mutated miR-744 binding sequences in the 3′-untranslated region (3′-UTR) of Notch1. (B) SW480 and HCT116 cells were cotransfected with miR-744 mimic or miR-NC and p-MIR-WT-Notch1-3′-UTR or p-MIR-MUT-Notch1-3′-UTR. At 48 h after transfection, cells were harvested and subjected to the analysis of luciferase activity using the Dual-Luciferase Reporter Assay System. *p < 0.05 versus miR-NC. miR-744 mimics or miR-NC was transfected into SW480 and HCT116 cells. (C) RT-qPCR and (D) Western blot analysis were applied to measure Notch1 mRNA and protein levels, respectively. *p < 0.05 versus miR-NC.

Fig 3: PAK1 deficiency exacerbates oxidative stress–related DNA damage and stress kinases, and drives Notch1 activation. (A) Immunohistochemistry (IHC) of γH2AX marking double-strand breaks. PAK1 deletion resulted in increased double-strand breaks. IHC of p65 indicating NF-κB activation and corresponding IRS. PAK1 deletion attenuated NF-κB signaling. (B) Western blot of colon samples from IL10 KO and DKO mice. In DKO mice, pERK signaling was abrogated, while blotting for pJNK showed increased dimerization, indicating pJNK activation. (C) Western blot of LBOs, which were treated with IL6 (10 ng/mL). Treatment resulted in activation of Notch1 and JNK in DKO, while ERK/AKT activation was impeded upon PAK1 loss. AKT, protein kinase B; γH2AX, phosphorylated H2A histone family member X; IRS, immune-reactivity score; pERK, phosphorylated extracellular signal-regulated kinase1/2; pJNK, phosphorylated c-Jun N-terminal kinase.

Fig 4: Restoration of Notch1 expression reverses the suppressive effects of miR-744 mimics in CRC cells. miR-744 mimic or miR-NC was introduced into SW480 and HCT116 cells in combination with pcDNA3.1 or pcDNA3.1-Notch1. (A) Western blot analysis was conducted at 72 h posttransfection to determine Notch1 protein level. *p < 0.05 versus miR-NC. #p < 0.05 versus miR-744 mimic < pcDNA3.1-Notch1. (B) CCK-8 and (C) Matrigel invasion assays were used to detect the proliferation and invasion of the above-mentioned treated cells. *p < 0.05 versus miR-NC. #p < 0.05 versus miR-744 mimic < pcDNA3.1-Notch1.

Fig 5: 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.