5 mpn

The human breast carcinoma cells MDA-MB-231, T47D and human umbilical venin endothelial cells (HUVEC) were obtained from Core Technology Facility of Center for Excellence in Molecular Cell Science, CAS. The MDA-MB-231 and T47D cells were cultured in high glucose Dulbecco's modified Eagle's medium (06-1055-57-1ACS, DMEM, Biological Industries, Israel) containing 10% fetal bovine serum (04-001-1ACS, Biological Industries, Israel) and 100U/ml penicillin/streptomycin (SV30010, Hyclone, Logan, UT). The HUVEC were cultured in RPMI 1640 medium (01-100-1ACS, Biological Industries, Israel) containing 10% fetal bovine serum (04-001-1ACS, Biological Industries, Israel) and 100 U/ml penicillin/streptomycin (SV30010, Hyclone, Logan, UT). Cells were cultured at 37°C in a humidified atmosphere with 5% CO₂.
The MDA-MB-231 and T47D cells were treated either with 10 μM PDTC (S3633, Selleck), 8nM AZD3965 (S7339, Selleck), or with 5 μM 5-MPN (S656801, Sigma-Aldrich) 24hrs for Western blot.
The HUVEC in 6-well plates were either transfected with a siRNA targeting STAT5A (s13535, Ambion), STAT5B (s13539, Ambion) or non-silencing control using Dharmafect1 (T-2001-03, Dharmacon) transfection reagent according to the manufacturer's protocol, or treated with anti-IL-6 (1:1000, WL02841, Wanleibio) 24hrs for Western blot.

GSCs or HEK293T cells were seeded in 96-well plates at a density of 10,000 or 3000 cells per well respectively. 5MPN (Sigma Aldrich) was diluted in DMSO and applied at various concentrations. Viability was measured after 48 hours using the CellTiter-Glo assay (Promega), according to the manufacturer’s protocol and using a Mithras LB 940 plate reader (Berthold Technologies). IC₅₀ values were determined by applying a four parameter logistic curve using Graphpad Prism 8.