Apoptosis Evaluation in HMEC-1 Cells

Twenty-four hours before treatment, HMEC-1 cells were plated in 6-well plates. Propidium iodide (PI) staining and flow cytometry analysis for apoptotic DNA-fragmentation (subG1 population) was performed 48, 72, or 96 h post-treatment. HMEC-1 cells were incubated for 15–30 min at RT with a staining solution (0.1 M Tris, 0.1 M NaCl, 5 mM MgCl2, 0.05% Triton X-100 (all Roth, Karlsruhe, Germany)), 62 µg/mL of RNaseA (AppliChem, Darmstadt, Germany), and 40 µg/mL PI (Sigma-Aldrich, St. Louis, MI, USA) [32 (link)]. Subsequently, samples were analyzed by flow cytometry (FACS Calibur, Becton Dickinson, Heidelberg, Germany; FL-2) [33 (link)]. Cell cycle phase distribution was analyzed with Kaluza software Version 2.1 to identify the subG1 population (apoptotic DNA-fragmentation, whole population), and, in a second step, the living cell population (G1, S, G2/M phase) was investigated for a G2/M arrest [15 (link)]. Statistical analysis was performed in GraphPadPrism Version 8.3.0. Exemplary histograms of flow cytometry are shown in Supplementary Figure S9.