Journal: iScience

Article Title: B3GNT5 is a novel marker correlated with malignant phenotype and poor outcome in pancreatic cancer

doi: 10.1016/j.isci.2024.110889

Figure Lengend Snippet: STAT5B promotes transcriptional activity of B3GNT5 in pancreatic cancer (A) CFPAC-1 and CAPAN-1 cells were transfected with the STAT5B overexpression vector and its controls, the transfection efficiency was verified by western blot after 48 h. (B) Quantitative real-time PCR and western blot analysis of B3GNT5 expression in tumor cells with or without STAT5B knockdown or overexpression. ∗∗ p < 0.01, compared with the Mock group. (C) The schematic diagram of positions and sequences of the predicted binding sites on the promoter of B3GNT5. The promoter sequence of B3GNT5 was downloaded from the UCSC website ( https://www.genome.ucsc.edu/ ) and the transcriptional regulation sites of B3GNT5 by STAT5B were predicted by the HumanTFDB database ( http://bioinfo.life.hust.edu.cn/HumanTFDB ). The dual-luciferase assay was performed 48 h after transfection with the indicated B3GNT5 promoter fragments and STAT5B overexpression vector or empty vector. (D) STAT5B overexpression plasmid was transfected into B3GNT5-stabilized knockdown tumor cells, and cell proliferation of respective cells was measured by CCK8 assay. ∗ p < 0.05, ∗∗ p < 0.01, compared with the control group; ∗∗ p < 0.01, compared with the STAT5B group. (E) The STAT5B overexpression plasmid was transfected into B3GNT5-stabilized knockdown tumor cells, which were further treated with 10 nM GEM after 24 h, and the percentage of apoptotic cells was evaluated by flow cytometry with annexin V-FITC/PI staining. Mean ± SD, N = 3. GEM, Gemcitabine.

Article Snippet: The primary antibodies including B3GNT5 antibody (1:500, cat# 20422-1-AP, Proteintech, China), STAT5B antibody (1:1000, cat# DF6078,Affinity, China), CD44 antibody (1:500, cat# DF63192, Affinity, China), CD326 antibody (1:500, cat# DF6311, Affinity, China), CxCR4 antibody (1:1000, cat# AF5279, Affinity, China), p-ERK1/2 antibody (1:5000, cat# AF1015, Affinity, China), ERK antibody (1:5000, cat# AF0155, Affinity, China), P-AKT antibody (1:5000, cat# AF0016, Affinity, China), AKT antibody (1: 5000, cat# AF6261, Affinity, China).

Techniques: Activity Assay, Transfection, Over Expression, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Binding Assay, Sequencing, Luciferase, CCK-8 Assay, Control, Flow Cytometry, Staining

Journal: iScience

Article Title: B3GNT5 is a novel marker correlated with malignant phenotype and poor outcome in pancreatic cancer

doi: 10.1016/j.isci.2024.110889

Figure Lengend Snippet:

Article Snippet: The primary antibodies including B3GNT5 antibody (1:500, cat# 20422-1-AP, Proteintech, China), STAT5B antibody (1:1000, cat# DF6078,Affinity, China), CD44 antibody (1:500, cat# DF63192, Affinity, China), CD326 antibody (1:500, cat# DF6311, Affinity, China), CxCR4 antibody (1:1000, cat# AF5279, Affinity, China), p-ERK1/2 antibody (1:5000, cat# AF1015, Affinity, China), ERK antibody (1:5000, cat# AF0155, Affinity, China), P-AKT antibody (1:5000, cat# AF0016, Affinity, China), AKT antibody (1: 5000, cat# AF6261, Affinity, China).

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, CCK-8 Assay, In Situ, Luciferase, Generated, Software, Flow Cytometry