Prolonged exposure to pyranoxanthone 2 (PX2) leads to apoptotic cell death. (a) TUNEL staining showing accumulation of apoptotic cells (green) in cells treated with the compound for 48 h. DNA was counterstained with DAPI (blue). Bar, 5 µm. (b) Apoptotic index in control cells and upon 48 h compound treatment, expressed as a percentage of total cells with statistical relevance of ** p < 0.01 by unpaired t-test. (c,d) Flow cytometry analysis of apoptosis by Annexin V/PI co-staining, 16 and 24 h after PX2 treatment. (c) Representative cytogram and (d) quantification of Annexin V-positive cells are shown with statistical relevance of * p < 0.05 by unpaired t-test. The quadrants Q were defined as Q1 = live (Annexin V- and PI-negative), Q2 = early stage of apoptosis (Annexin V-positive/PI-negative), Q3 = late stage of apoptosis (Annexin V- and PI-positive) and Q4 = necrosis (Annexin V-negative/PI-positive).