No 1.5 22 22 mm coverslips

The In Situ Cell Death Detection Kit, Fluorescence (Roche, 11684795910) was used to label quantify DNA strand breaks with TUNEL according to the manufactures protocol. Cells were seeded in 6-well plates with no. 1.5 22×22 mm coverslips (Electron Microscopy Sciences, 72204–01) at 1.5×10⁵ cells/well, grown for 48 h, the washed with 1x PBS, and treated with complete or serum-free media for 18–24 h. After incubation, media was removed and cells were washed 3x with 1x PBS, fixed in 4% PFA (ThermoFisher, J19943.K2) for 10 min, washed 3x with 1x PBS, and stored in 1x PBS at 4°C until use. After TUNEL, immunostaining was performed. Samples were washed 3x with 0.02% Tween20 (Sigma-Aldrich, P1379) in 1x PBS for 15 min each, blocked using 3% BSA/10% FBS/0.02% Tween20 in 1x PBS for 3 h, and incubated with primary antibody for Cleaved Caspase-3 (1:200, Cell Signaling Technology) overnight at 4°C. Samples were then washed 3x for 15 min each with 0.02% Tween20, goat anti-rabbit AlexFluor594 (1:200, Invitrogen) in blocking solution was added for 2 h at room temperature, and washed 3x for 15 min each with 0.02% Tween20. Coverslips were mounted using Vectashield with DAPI (Vector, H-1200) microscope slides and sealed with nail polish. Analysis was performed in ImageJ using the Particle Analyzer plugin. Apoptotic cells were identified as cells positive for both TUNEL and Cleaved Caspase 3.

DNA synthesis was directly measured in live cells with the EdU Staining Proliferation kit iFluor 488 (Abcam, ab219801) following the manufacture protocol. Cells were seeded in 6-well plates with no. 1.5 22×22 mm coverslips (Electron Microscopy Sciences, 72204–01) at 1.5×10⁵ cells/well, cultured for 48 h, then washed with 1x PBS, and treated with complete or serum-free media. After 20–24 h incubation, cells were incubated with 10 μm EdU solution for 4 h, fixed in 4% paraformaldehyde (PFA; ThermoFisher, J19943.K2) for 10 min, permeabilized, and labeled using kit components. Coverslips were mounted using Vectashield with DAPI (Vector, H-1200) microscope slides and sealed with nail polish. Analysis was performed in ImageJ using the Particle Analyzer plugin and proliferation was calculated as the fraction of EdU positive cells.