NT157 has antineoplastic effects and inhibits IRS1/2 and STAT3/5 in JAK2V617F-positive myeloproliferative neoplasm cells

摘要

Cell viability was determined by methylthiazole tetrazolium (MTT) assays for HEL cells with or without NT157 treatment (0.2, 0.4, 0.8, 1.6, and 3.2 µM) for 24, 48, and 72 h. Bar graphs represent the mean ± SD of at least three independent experiments, and the dots represent the value of each experiment; ***  b Apoptosis was detected by flow cytometry in HEL cells with or without NT157 treatment (0.2, 0.4, 0.8, and1.6 µM) for 24, 48, and 72 h using an annexin V/PI staining method. Bar graphs represent the mean ± SD of at least three independent experiments where annexin V-positive cells were quantified, and the dots represent the value of each experiment; **  p c Representative flow cytometry dot plots are shown for each condition; the upper and lower right quadrants cumulatively contain the apoptotic population (annexin V-positive cells). Caspases 3, 8, and 9 levels (total and cleaved) were assessed by western blot analysis in total cell extracts from HEL with NT157 treatment (Ø, 0.2, 0.4, 0.8, 1.6, and 3.2 µM) for 24 h; membranes were reprobed with antibodies to detect total target protein and/or actin, and then they were developed with a SuperSignal™ West Dura Extended Duration Substrate system and a Gel Doc XR + system. Ki-67 mean fluorescence intensity (MFI) was determined by flow cytometry after incubation of HEL cells with NT157 (Ø, 0.2, 0.4, 0.8, 1.6, and 3.2 µM) for 24 h; bar graphs represent the Ki-67 MFI normalized to the respective untreated control cells, and the results are shown as the mean ± SD of three independent experiments. The dots represent the value of each experiment; **  p f Histograms show Ki-67 MFI peaks for all tested conditions. Each condition is represented by one color that is defined in the legend located in the left panel. Colonies containing viable cells were detected by MTT assay after 10 days of culture with NT157, and data are normalized to the corresponding untreated control. Colony images are shown for one experiment, and the bar graphs show the mean ± SD of five independent experiments. The dots represent the value of each experiment; *  h Cell cycle progression was determined by a BD Cycletest™ Plus DNA Reagent Kit in HEL cells treated with NT157 (Ø, 0.2, 0.4, and 0.8 µM) for 24 h. A representative histogram for each condition is illustrated; cell cycle phases are detected based on DNA quantity: blue (cells in G /G ), green (cells in S), and pink (G /M cells). Bar graphs represent the mean ± SD of the percent of cells in the G /G , S, and G /M phase of three independent experiments; **  p