Journal: Molecular cancer therapeutics

Article Title: Anti-Leukemia Effects of Notch-Mediated Inhibition of Oncogenic PLK1 in B-Cell Acute Lymphoblastic Leukemia

doi: 10.1158/1535-7163.MCT-18-0706

Figure Lengend Snippet: (A) Percentage of Notch 1-4 receptor-positive cells on B-ALL patient samples and a representative cell line was determined by flow cytometry as the fraction of cells exceeding the intensity of the corresponding isotype control. Patients are represented by the same symbol for each receptor with PH-ve and PH-like subtypes discriminated by green and blue, respectively. Mean percentage for each receptor is represented by the horizontal bar. (B) B-ALL patient samples (n=3) were cultured on plate-bound human DLL1-Fc, DLL3-Fc, DLL4-Fc, Jagged1-Fc, Jagged2-Fc, and an IgG-Fc control. Viability was determined every 24 hours as the percentage of cells PI positive by flow cytometry. (C) Cell lines representing T-ALL (CEM) and B-ALL (SB, JM1, Nalm6) and samples from Ph-like B-ALL patients (Pt.B1, Pt.B2) were co-cultured on human bone marrow stromal HS5 cells expressing GFP (control) or DLL1. Viability was determined every 24 hours by trypan blue (n=3). (D) Representative histograms of Annexin V positivity in the same cells co-cultured with HS5 cells expressing DLL1 or control at 96 hours. (E) RNA expression of Notch downstream genes HES1, DTX1, and HEY1 in B-ALL patient samples (n=3) co-cultured for 24 hours on HS5 cells expressing GFP or DLL1; with and without a DLL1 blocking antibody, was assessed by qRT-PCR. (F) A representative western blot from the same experiment depicting HES1(abcam ab) at the protein level.

Article Snippet: Plates were treated with 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO), washed with phosphate-buffered saline solution (PBS), and incubated with 1 μg of human ligands fused to IgG Fc domains, i.e., DLL1-Fc (ALX-201-765-0025; Enzo Life Sciences, Farmingdale, NY), DLL3-Fc, DLL4-Fc (Adipogen), Jagged-1Fc or −2Fc (1277-JG-050 and 1726-JG-050, R &D Systems, Minneapolis, MN) or control IgG-Fc (Jackson Laboratory, Bar Harbor, ME) in 0.1% BSA independently, with rocking, overnight at 4°C.

Techniques: Flow Cytometry, Cell Culture, Expressing, RNA Expression, Blocking Assay, Quantitative RT-PCR, Western Blot

Journal: Oncotarget

Article Title: Asiatic acid attenuates lipopolysaccharide-induced injury by suppressing activation of the Notch signaling pathway

doi: 10.18632/oncotarget.24542

Figure Lengend Snippet: ( A ) Cells were treated with various concentrations of AA in the absence or presence of LPS for 48 h, followed by analysis of Notch receptors and Dll4 expression by PCR. ( B ) Protein expression of Notch3 and Hes1 in AA and LPS treatment of Raw264.7 cells. ( C ) RAW264.7 cells were cultured in the presence of LPS (0.1 μg/mL) in addition to for 48 h. IL-1β and IL-6 expression was determined by qPCR. ( D ) RAW264.7 cells were cultured in the presence of LPS (0.1 μg/mL) in addition to Dll4 fusion protein (1 μg/mL) for 48 h. IL-1β and IL-6 expression were determined by qPCR. ( E ) Association of Notch3 and IL-6 gene promoter region was measured in the immunoprecipitate by quantitative PCR relative to that for input DNA. * p < 0.05 versus control, # p < 0.05 versus LPS control. Data are shown as mean ± SD ( n = 5), and each experiments were duplicated for 3 times.

Article Snippet: Recombinant mouse Dll4 protein (2.5 μg; R&D) or 2.5 μg IgG (Santa Cruz Biotechnology) as a control were diluted in 1 mL 0.1% gelatin in phosphate-buffered saline.

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction