Fig 1: Downregulation of NUCB1 was associated with poor prognosis in PDAC patients. (A–D) The association between gene expression patterns and survival rates of patients with PDAC was analyzed using the UALCAN database (annotated as PAAD dataset) (http://ualcan.path.uab.edu/analysis.html). Four members of the GEF family (CCDC88A, CCDC88C, NUCB1, and NUCB2) were examined. (E) Quantitative PCR was used to detect NUCB1 expression in 25 pairs of pancreatic cancer and adjacent tissues. (F) Immunohistochemistry (IHC) was used to examine NUCB1 protein expression in tissues. Scale bar: 100 µm. (G,H) Patients were divided into two groups based on IHC staining for survival curve analysis (G) and multivariate regression analysis (H).
Fig 2: NUCB1 overexpression suppressed cell proliferation and increased the anti-tumor effects of GEM. (A–C) SW1990 (A) and CFPAC1 cells (B) were infected with virus expressing NUCB1 (oeNUCB1) or control vector (oeNC), and BXPC-3 cells (C) were infected with shRNAs targeting NUCB1 (shNU-1, shNU-2) or control shRNA (shNC). Western blot was conducted to determine protein levels. (D–F) CCK-8 assay was performed to assess proliferation. (G–I) SW1990, CFPAC1 and BXPC-3 cells were treated with 0, 5, 20, and 50 µM GEM for 24 h (0 µM represents cells treated with vehicle, DMSO) and apoptosis was detected. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control (shNC or vector).
Fig 3: Proposed Model of Regulation. NUCB1 regulates ATF activity to modulate UPR and autophagy. METTL3 promotes m6A modification of NUCB1 via YTHDF2.
Fig 4: NUCB1 suppressed GEM-induced UPR and autophagy. SW1990 cells overexpressing NUCB1 were treated with 20 µM GEM. (A,B) Western blot was conducted to determine the proteins levels of NUCB1, UPR-associated genes (GRP78, CHOP, p90ATF6, p50ATF6) and autophagy-associated genes (p62, LC3-II/-I, and XBP1). GAPDH was used as a loading control. (C) Immunofluorescence staining was performed to detect LC3 expression. Scale bar: 50 µm. *p < 0.05, ***p < 0.01, ***p < 0.001.
Fig 5: ATF6 reversed the effects of NUCB1. SW1990 cells simultaneously overexpressing ATF6-pATF6 (active) and NUCB1 were treated with 20 µM GEM. Western blot (A,B) was conducted to determine protein levels of GRP78, CHOP, p90ATF6, p50ATF (active), NUCB1, p62, LC3-II/-I, and XBP-1. Immunofluorescence staining (C) was performed to detect LC3 expression. Scale bar: 50 µm. *p < 0.05, **p < 0.01.
Supplier Page from Abcam for Anti-NUCB1 antibody