Fig. 4

NOTCH1 is a functional target of YTHDF1 in promoting stemness and tumorigenesis. a Ythdf1 conditional knock-in activated the NOTCH signaling pathway at the mRNA level (N = 3 per group). b NOTCH1, HES1, HEY1, p-H2AX, and H2AX protein expression in the Ythdf1 conditional knock-in mouse model. c NOTCH1 protein expression in YTHDF1-overexpressing CSCs. d Representative images of immunofluorescence co-staining of YTHDF1 and NOTCH1 in control and YTHDF1-overexpressing PDO816 cells. Scale bar = 100 μm. e Effect of siNOTCH1 in the context of YTHDF1 overexpression on CSC28 and LS174TS self-renewal capacity (seeded at 1, 2, 5, and 10 cells per well for 7 days) and f spheroid formation ability (n = 5 per group). Scale bar = 20 μm. g Effect of DAPT in YTHDF1-overexpressing cells on CSC28 and LS174TS self-renewal capacity (seeded at 1, 2, 5, and 10 cells per well, 7 days) and h spheroid formation ability (n = 5 per group). Scale bar = 20 μm. i Schematic of intestinal tumor organoid construction. j Effect of DAPT on Apc^Min/+ and Apc^Min/+Ythdf1^cKi organoid formation (left and middle) and secondary organoid formation ability (right) (N = 5 per group). Scale bar = 20 μm. k Experimental schematic of Ythdf1^lslLgr5-Cre in the AOM/DSS^-induced CRC model (left). Representative images of colonoscopy (right). l Representative images of the colon (left). Tumor number and load in WT (N = 7) and Ythdf1^cKi (N = 5) mice and in DAPT-treated WT (N = 17) and Ythdf1^cKi (N = 11) mice (right). m Ki67 staining of colon tissues from littermate WT (n = 12) and Ythdf1^cKi (n = 12) mice. Scale bar = 25 μm. Spheroid and organoid size quantifications were performed via ImageJ. The data are presented as the means ± SDs. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ordinary one-way ANOVA (a); two-way ANOVA (f, h, j, l, m); ELDA: extreme limiting dilution analysis (e, g)