Brdu incorporation assay

Cell proliferation was measured by BrdU Incorporation assay (Promega, WI, USA) and apoptosis by Caspase-ApoGlo 3/7 assay (Promega, WI, USA). The impact of miR-24-3p on capillary-like cord formation was prompted in vitro using a Matrigel assay. HUVECs were transfected with pre-miR-24-3p or anti-miR-24-3p and control or infected with Ad. Decoy-miR-24-3p or Ad. Null (control). Next, cells were seeded in 96-well plates previously coated with growth factors-enriched Matrigel (Matrigel Matrix, Basement Membrane, BD, Oxford, UK). Endothelial network formation was quantified by morphometric analysis. Photomicrographs (40X) were taken 12 h after the start of the experiment: three photomicrographs were taken for each treatment, and each treatment was performed in triplicate. ImageJ was used to quantify the total length of the capillary-like structures. For cell migration assay, HUVECs were transfected with either anti-miR-24-3p or scramble control and seeded in a 12-wells plate to reach confluence by 48h. Then, in order to inhibit cell proliferation, cells were incubated with 2mM of hydroxyurea, and the monolayer was scratched. Images were taken at the beginning of the experiment (time zero, T0), 5, and 12 h. Five photomicrographs were taken for each treatment, and each treatment was performed in triplicate.

The apoptosis of the GBM cells was analysed by Hoechst 33258 staining (Invitrogen) as previously described.²² Alternatively, the apoptotic cells were also analysed by annexin‐V/PI staining (Thermo Fisher) followed by flow cytometry (BD accuri C6, BD Biosciences) analysis as previously described.²²The cell viability was determined by MTT assay as described in our previous study.²¹ Proliferation was analysed using BrdU incorporation assay (Promega) in accordance with the manufacturer's protocol. Cells were normalized by a standard curve.