Flow cytometry analysis of HeLa cells stained with FITC-Annexin V and PI and Western blotting of enhanced expression of cleaved caspase-3. Flow cytometry analysis of FITC-Annexin V and PI stained HeLa cells with untreated HeLa cells (A) and HeLa cells treated with 0.5 mg/ml P. indica root extract for 48 hours (B). In each scatter plot, upper-left quadrant (Q1-1) shows naked nucleus cell mass, upper-right quadrant (Q2-1) shows necrotic cell mass, lower-left quadrant (Q3-1) shows survival cell mass, and lower-right (Q4-1) shows apoptotic cell mass. In control cells, there were only 0.1% of apoptotic cells (Figure 7A; Q4-1). After 48 hours of treatment with 0.5 mg/ml P. indica root aqueous extract, approximately 43% of cells were detected as undergoing necrosis and apoptosis (Figure 7B; Q2-1+Q4-1). (C) A representative of Western blotting of time-dependent increasing expression of cleaved caspase-3. Cleaved Caspase-3 (Asp175) monoclonal antibody (5A1E; Cell Signaling Technology) used detects levels of the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175. Lane 1: untreated; lane 2: 1 day of 0.5 mg/ml extract treatment; lane 3: 2 days of 0.5 mg/ml extract treatment; lane 4: 3 days of 0.5 mg/ml extract treatment. Each experiment was done in triplicate.