A total of 50,000 cells per well were plated in triplicate in a 12 well plate. Cells were then irradiated with varying doses of radiation. On day 2, cell viability was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega). Viability was normalized to the untreated control for each condition and graphed as a percentage of the total.
For post-radiation apoptosis assays, cell were irradiated and when indicated, cathepsin L inhibitors E64D (Enzo, #PI107–0001) and Z-FY-CHO (Med Chem Express, #HY-128140) were added immediately after radiation. Cells were incubated for 24 h, then treated with Accutase (Biolegend, #42320) to ensure a single-cell suspension and stained with annexin V and propidium iodide as described below. Double positive cells were quantified. A similar experiment was performed with Temozolomide (Santa Cruz, #CAS 85622–93-1) as a replacement for irradiation.
For post-radiation apoptosis assays, cell were irradiated and when indicated, cathepsin L inhibitors E64D (Enzo, #PI107–0001) and Z-FY-CHO (Med Chem Express, #HY-128140) were added immediately after radiation. Cells were incubated for 24 h, then treated with Accutase (Biolegend, #42320) to ensure a single-cell suspension and stained with annexin V and propidium iodide as described below. Double positive cells were quantified. A similar experiment was performed with Temozolomide (Santa Cruz, #CAS 85622–93-1) as a replacement for irradiation.