Fig 2: DIOS downregulates Notch3 signaling and inhibits the NF-κB pathway by inactivation of its upstream regulator IKKα/IKKβ. (A) Notch3, cleaved Notch3 and NF-κB were detected by immunoblotting. (B) The p53 cellular location and protein level was analyzed by immunofluorescence (magnification, ×200). (C) IKKα, IKKβ and NF-κB protein level alterations were determined following DIOS stimulation. DIOS, diosmetin; NF, nuclear factor; IKK, IκB kinase.

Fig 3: NOTCH3 knockdown exerts anti-oncogenic effects in vitro.A NOTCH3 is overexpressed in all ten GC cell lines compared with normal gastric tissues. B The mRNA and protein expression of NOTCH3 after siRNA-mediated knockdown in AGS and MKN28 cells. C NOTCH3 knockdown by two siRNAs suppressed AGS and MKN28 cell proliferation (*P < 0.05; **P < 0.001). D NOTCH3 silencing decreased monolayer colony formation ability of the cancer cells (**P < 0.001). E NOTCH3 deletion inhibited cell invasion ability of GC cells (**P < 0.001). F Western blot analysis demonstrated the activation of cleaved caspase-3 and cleaved-PARP induced by NOTCH3 knockdown in all four GC cells. G Flow cytometry for apoptosis analysis of siScramble and siNOTCH3 transfectants. The representative bar chart of cell distribution was selected from two independent experiments with the same results (**P < 0.001). H Enrichment plots of apoptotic gene expression signatures according to NOTCH3 expression in NCBI/GEO/GSE57303 cohort (upper panel, APOPTOSIS_GO, P = 0.039; lower panel, KEGG_APOPTOSIS, P = 0.010). The barcode plot indicated the position of the genes in each gene set; red and blue colors represented the high and low expression of NOTCH3, respectively. ES enrichment score, NES normalized enrichment score. I The immunohistochemistry images of NOTCH3 in primary GC samples. NOTCH3 was detected both in the cytoplasm and nuclei of the intestinal and diffuse-type cancer cells. J NOTCH3 nuclear accumulation was associated with poor disease-specific survival (P = 0.0018).

Fig 4: NOTCH paralogues induce a mesenchymal phenotype in ADRN neuroblastoma cells. a Western blot analysis of cell lysates of SH-SY5Y with inducible overexpression of NOTCH1-IC, NOTCH2-ICFLAG or NOTCH3-IC. Immunoblots show NOTCH1-IC, NOTCH2-ICFLAG or NOTCH3 proteins as well as protein of the known NOTCH target gene HES1 to confirm induction of each transgene. Lysates are analyzed for MES-markers (FN1 and SNAI2) and ADRN-markers (PHOX2A, PHOX2B, and DBH). β-actin was used as loading control. IC intracellular domain, TM transmembrane domain, dox doxycycline. Source data are provided as a Source Data file. b Transwell migration assay of SH-SY5Y cells with inducible overexpression of NOTCH1-IC, NOTCH2-ICFLAG or NOTCH3-IC. Cells were allowed to migrate for 48 h. dox, doxycycline. Box plots show the number of migrated cells per High-Power Field (HPF). Whiskers denote the interval within 1.5 times the interquartile range (box edges) of the median (center line). Two-sided Student’s t-test assuming equal variance was used to calculate statistical difference, ***p < 0.001. Source data are provided as a Source Data file

Fig 5: Increased RhoA/Rho kinase activity and blunted [Ca2+]i transients in CADASIL VSMCs.(A) VSMC Rho kinase activity assessed by ELISA (n = 4–5; Student’s t test). (B and C) Expression of ROCK2 (B) and ROCK1 (C) in CADASIL and control VSMCs in the absence and presence of a γ-secretase inhibitor (GSI). Upper panels: Representative immunoblots. Lower panels: Quantification of ROCK2 and ROCK1 protein expression normalized to α-tubulin (n = 6–10; two-way ANOVA with Bonferroni’s post hoc test). (D) Calcium transients were measured by live cell fluorescence imaging using the fluoroprobe Cal-520 AM. Representative tracings of VSMC [Ca2+]i responses to Ang II (1 × 10–7 mol/L) in CADASIL and control groups. Experiments were repeated 4 times/group with greater than 30 cells studied/field. (E) [Ca2+]i calculated as the area under the curve. Arrow indicates time of Ang II stimulation (n = 4; Student’s t test). Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01 versus control; #P < 0.05 versus vehicle plus CADASIL.