Annexin 5 fitc and 7 aad

U87 and U251 cells were seeded in six-well plates (300,000 cells/well) 24 h prior to treatment. Each well was treated with various concentrations of 28, ent-28, TMZ, or vehicle control DMSO for 24 h. DMSO was used as a negative control. The cells were then washed and stained with Annexin V-FITC and 7-AAD (BioLegend), according to the manufacturer’s instructions, and were analyzed on a MACSQuant^® Analyzer 10 (Miltenyi Biotec Inc.). Staurosporine (STS) treatment at 1 μM served as a positive control for Annexin V staining and DMSO was used as a negative control. The files were converted to FCS compatible format and analyzed using FlowJo analysis software (Treestar). The quantitative analysis of the percentage of apoptotic cells was reported from three independent experiments.

5 x 10⁵ cells were incubated with 1 µg/ml of the indicated antibody for 20 h. Subsequently, cells were washed twice with PBS supplemented with 1% BSA and 0.1% NaN₃ and incubated with Annexin V-FITC and 7-AAD from BioLegend (cat. no. 640922) according to manufacturer’s introductions. Samples were measured on a Navios flow cytometer and programmed cell death (PCD) was calculated as percentage of Annexin V⁺ cells representing early and late apoptotic cells.

Palbociclib (PD0332991) HCl (catalog no. S1116) was purchased from Selleckchem (www.selleckchem.com). The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (CellTiter 96^® Non-Radioactive Cell Proliferation Assay; Promega, Madison, WI, USA) was utilized to assess the effect of palbociclib on cell viability. Cells were seeded in culture media (3 × 10⁴ cells/well) in triplicate in a 96-well plate and incubated overnight. Palbociclib was added at eleven different concentrations (0, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40, and 80 µM) at 37 °C. After 48 h incubation, the cells were washed with fresh media and were grown in palbociclib-free media for an additional 24 h, until MTT assays were done as specified by the manufacturer’s instructions. IC₅₀, a concentration at which cell viability was down to half, was calculated using Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA).
For apoptosis assays, 10⁶ cells were initially plated on 6-well plates and incubated overnight. Cells were treated with 5 µM of palbociclib, incubated for 24 h, and subjected to apoptosis assay with flow cytometry by using Annexin V-FITC and 7-AAD (catalog no. 640922; BioLegend, San Diego, CA, USA). All experiments were independently repeated in quadruplicate.