Fig. 3: ZFYVE28 was transcriptionally regulated by NOTCH via RBP-Jκ.

a–d GSEA of the liver transcriptome sequencing results showed that the Notch pathway was suppressed in insulin-sensitive obese mice in the HFD-4w group (a, b) but activated in insulin-resistant mice in the HFD-12w group (c, d); n = 3 biologically independent samples per group. The P values in GSEA are calculated empirically with permutation test by permuting the gene labels at random according to the GSEA algorithm, and FDR q values (corrected P values) are calculated by Benjamini–Hochberg FDR correction. The NES and FDR q values are shown in the images. NES normalized enrichment score, FDR false positive rate, GSEA gene set enrichment analysis. e–h Western blotting analysis of Notch1 protein in livers from HFD-4w group mice (e) and HFD-12w group mice (f). The quantitative analysis results (g, h) are shown; n = 3 biologically independent samples per group. i Relative mRNA expression of HES1, HEY1 and ZFYVE28 in HepG2 cells treated with insulin at 10⁻⁶ M; n = 6 biologically independent samples per group. j Percentage mRNA remaining of ZFYVE28 in HepG2 cells treated with ActD in the presence or absence of insulin stimulation at 10⁻⁶ M; n = 3 biologically independent samples per group. k Representative western blot results of NICD in HepG2 cells treated with insulin; the quantitative analysis results of three independent parallel experiments are shown. l, m The expression levels of HES1, HEY1 and ZFYVE28 following NOTCH1 overexpression (l) and NOTCH1 knockdown (m) in HepG2 cells; n = 3 biologically independent samples per group. n RBP-Jk binding site prediction in the ZFYVE28 promoter region using JASPAR. o Chromosome immunoprecipitation assay showed the fold enrichment of RBP-Jk in the ZFYVE28 promoter region; n = 3 biologically independent samples per group. p, q Diagram of the dual luciferase reporter system (q) and results of the luciferase activity assays (p); n = 5 biologically independent samples per group. r, s Representative western blotting results of NICD levels (r) in HepG2 cells treated with insulin and KRA-533. The quantitative analysis results of three independent parallel experiments (s) are shown. t–w Representative western blotting results of Nicd levels, as well as quantitative analysis results of three independent parallel experiments, in primary hepatocytes from WT mice (t, u) and insulin-resistant mice (v, w). Data are shown as means ± SD, and P values are determined by unpaired two-tailed Student’s t-test (g–j, l, m, o, p, s, u, w) or one-way ANOVA followed by Tukey’s post hoc tests (k). The exact P values are shown in the figure. Source data are provided as a Source Data file.