Fig 2: Examination of genes related to BCAA and fatty acid metabolism in PDAC tissue. A–F: Data mining in GSE62165 dataset. (A) The expression level of SLC carriers (SLC7A5, SLC1A5, SLC3A2, SLC43A1 and SLC43A2) between PDAC and normal tissue. (B) GSEA analysis revealed enriched MYC and NOTCH signaling pathway in PDAC tissue. (C) The expression level of MYC and NOTCH (NOTCH1-4) between PDAC and normal tissue. (D) BCAA catabolism related genes BCAT1, BCAT2, BCKDHA, BCKDHB, BCKDK, ACADS and ACADSB were found significantly down-regulated in PDAC group. (E) The expression of lipogenesis related enzymes including FAS, ACACA and ACLY were significantly up-regulated in PDAC tissue. (F) CPT1A, the rate-limiting enzyme for fatty acid oxidation was notably upregulated in PDAC tissue. G–J: Data mining in GSE102238 dataset. (G) The expression level of SLC carriers (SLC7A5, SLC1A5, SLC3A2, SLC43A1 and SLC43A2) between PDAC and adjacent normal tissue. (H) NOTCH3 and NOTCH4 were upregulated in PDAC tissue while MYC was down-regulated. (I) Gene expression of BCAT1, BCAT2, BCKDHA, BCKDHB and ACADSB were decreased in PDAC tissue. (J) FAS and ACACA were increased in PDAC tissue.

Fig 3: Notch4 localization in breast cancer cell lines.a Representative confocal images of cell lines stained for Notch4 (green) or Nucleolin (red) and merged images. b Co-localization of Nucleolin (red) and Notch4 (green), quantified by Manders Coefficient (described in “Methods”) for the indicated cell lines. c, d Apoptotic nuclear damage in Hs578T cells pretreated with the indicated siRNA for 48 h and then cultured untreated (UT) or with 10 µM etoposide or 10 µM 5-FU for 24 h. Inset: percentage of mRNA levels in siRNA-transfected groups. e, f MDA-MB-231 cells treated with etoposide for 6 h were lysed and subjected to immunoprecipitation with antibody to Notch4 (e) or NCL (f) and IgG (isotype control). Immunoprecipitates were analyzed by western blotting for NCL, Notch4, NPM, FBL, Actin, and IgG. Data show the mean ± S.D. of three independent experiments. Scale bar: 5 µm.

Fig 4: The scheme of the present study. SOX2 regulates HEY1 and HEY1 creates a reciprocal loop with NOTCH4 to promote HNSCC EMT, self renewal and tumorigenicity.

Fig 5: Lentivirus mediated expression of Notch intracellular domain 1–4. A. Localization of the Notch receptors in differentiated human airway epithelium. Immunohistochemical staining of ALI day 28 sections for NOTCH1, NOTCH2, NOTCH3 and NOTCH4. Matched isotype IgG was used as a negative control. Scale bar 20 µm. Data shown are representative images of n = 3 independent experiments. B. Schematic of human Notch intracellular domains (NICD) of Notch receptor 1–4. All NICDs contain a RAM23 domain (RAM), ankyrin repeats (ANK), and proline, glutamic acid, serine, threonine-rich (PEST) sequences. NICD1, 2 and 3 contain a notch cytokine response (NCR) sequence immediately C-terminal of the ankyrin repeats that regulates functional activity. Further towards the C-terminus, NICD1 and NICD2 contain C-terminal transcriptional activation domains (TAD). Amino acid sequences describe the region for each Notch receptor that encompasses the NICD. C-F. Western analysis of NICD1-4 from lentivirus expression constructs. Lane 1—Mock (untransfected); lane 2—Lenti-GFP; and lane 3—Lenti-NICD. Shown is the expression of C. NICD1, D. NICD2, E. NICD3 and F. NICD4. GAPDH was used as a loading control.