Fig. 4. N1DARP inhibits pancreatic cancer initiation and progression by mitigating activation of the Notch signaling pathway.

a Left: RNA sequencing analysis using Capan1 transfected with vector or N1DARP overexpression plasmid. Right: GSEA analysis demonstrating the enrichment of differentially expressed genes in Notch, Hedgehog, PI3K/AKT, and JAK-STAT3 pathways. b Western blotting analysis showing inhibition of Notch1 signaling and its crosstalk with other pathways in Capan1 transfected with FLAG-tagged N1DARP plasmid in a dose-dependent manner. c Co-IP followed by mass spectrometry revealing the N1DARP–N1ICD interaction in HEK293T. d Immunofluorescence confocal microscopy showing the co-localization of N1DARP and N1ICD in the cytoplasm by the simultaneous elevation of fluorescence intensity of these two proteins in Panc1 cytoplasm. The red arrows indicate the co-localization site and the red line represents fluorescence intensity measured in the right panel. e In vitro pulldown assay performed using purified GST and GST-tagged N1ICD fusion protein incubated with Panc1 cell lysates, followed by western blotting analysis to detect the direct interaction between N1DARP and N1ICD. f Western blotting analysis showing the expression of key proteins in Notch signaling and its related pathways with N1DARP overexpression or repression, followed by treatment with Notch signaling activator JAG-1 or inhibitor DAPT. g, h Cell proliferation detected by EdU assay and stem cell properties measured by tumor sphere formation assay using Capan1 (g) transfected with N1DARP followed by treatment with JAG-1, a Notch pathway agonist, and Panc1 (h) with N1DARP knockout following DAPT treatment, a Notch pathway inhibitor. i RNA expression of Notch receptors (Notch1, Notch2, Notch3, and Notch4) and Notch signaling targeted genes (c-myc, HES1, and p21) measured by qRT-PCR using Capan1 with vector or N1DARP overexpression. j Immunohistochemistry analysis using tissue microarray from seventy-five pancreatic cancer patients detecting the correlation between N1DARP and N1ICD. k Western blotting analysis exhibiting N1ICD remaining level at indicated time in Capan1 and Panc1 with N1DARP overexpression or knockout and treatment with CHX, a protein synthesis inhibitor. l Western blotting analysis exhibiting N1ICD levels remaining at indicated time after treatment with Capan1 with N1DARP overexpression and BAF, a lysosome inhibitor, and MG132, a proteosome inhibitor. The gray level was quantified using ImageJ. The data are presented as the mean ± SD of three independent experiments. *P < 0.05 by Student’s t-test (g–i); P < 0.05 was considered statistically significant by Pearson’s correlation test (r, Pearson’s correlation coefficient) (j); ns no significance; *P < 0.05, ***P < 0.001 by one-way ANOVA (k, l). Scale bars, 10 μm (d), 50 μm (j).