Penicillin g and streptomycin sulfate

NIH3T3 and HEK-293T cells were maintained in DMEM containing high glucose, L-glutamine and sodium pyruvate (HyClone) supplemented with 10% (v/v) fetal bovine serum (HyClone), penicillin G and streptomycin sulfate (Invitrogen) in a humidified incubator at 37°C with 5% CO₂. Luciferase assays were performed using a pGL3T-Flt1 promoter reporter construct that was generated in our laboratory and has been previously described. For co-transfection and luciferase assays, NIH3T3 cells were grown in 24-well plates (70% confluence) and transfections were performed in triplicates with 250 ng/well of the pGL3T-Flt1 promoter reporter construct and different doses (50, 100, 200, 400 ng) of pcDNA-HA-Etv2 or pCMV-Vezf1-Flag-Myc (OriGene) expression plasmid using Lipofectamine 3,000 (Invitrogen) according to manufacturer’s instructions. Separately, the pGL3T-Flt1 construct was co-transfected into NIH3T3 cells with single dose of Etv2/Vezf1 and different doses of Vezf1/Etv2 expression plasmids. In all these co-transfection experiments, the total amount of DNA was made equal using pcDNA3 as balancing plasmids in different transfection mixtures. Cells were lysed and luciferase assays were performed using the Dual-Luciferase assay (Promega) following the directions outlined in the user manual.

A total of 8 human bone marrow-derived MSCs from 8 donors were purchased from ALLCELLS and Lonza at passage 1 (Additional file 1: Table S1). By flow cytometry, all MSCs revealed positive expression for CD29, CD44, CD73, CD90, CD105, and CD166 [31 (link), 38 (link)]. MSCs were expanded in medium containing alpha-MEM (Invitrogen), 10% FBS (Atlanta Biologics), 1% L-glutamine (Invitrogen), and 1% penicillin G and streptomycin sulfate (Invitrogen) under a humidified atmosphere of 5% CO₂ at 37 °C as previously reported [31 (link)]. Upon reaching 80% confluency, MSCs were detached from the flask using 0.25% trypsin/1 mM EDTA solution (Invitrogen) and replated at 60 cells/cm². MSCs were expanded to passage 7. Only two donor’s MSCs, 127756 and PCBM1655 did not reach passage 7. The second set of MSCs (n = 7) and mesoderm cells lines (n = 4) were expanded from passage 4 to 8 in marrow stromal cell growth medium from Cell Applications (San Diego, CA) as described in Bellayr et al. (2016) [37 (link)]. Cancer cell lines were purchased from ATCC (Manassas, VA) and cultured according to the manufacturer’s instructions.