Genistein promoted cell death of stressed cancer cells at sub-toxic concentration to unstressed cells. (a). Dose-dependent responsive curves of genistein on unstressed cells (G Alone) and stressed cells (E + G) of HeLa for 48 h treatment by MTT assay. Percentage of inhibition was calculated as: [(mean value of untreated control – each value of G alone group)/ mean value of untreated control] x 100% for Genistein Alone (G Alone) group; [(mean value of stressed + fresh medium (E + R) readings – each value of stressed + genistein (E + G) group)/ mean value of stressed + fresh medium (E + R) readings] x 100% for stressed + genistein (E + G) group. Two-way ANOVA was used to test the significance of ethanol stress as the source of variance. (b). LDH cytotoxicity assay of genistein treatment on HeLa cell for 18 h. Upper figure: LDH assay reading of dose-dependent genistein treatment: low control: untreated sample; High control: Triton X-100 treated. Bottom figure: percentage of cytotoxicity of dose-dependent genistein treatment. (c). BrdU cellular proliferation assay of dose-dependent genistein treatment of HeLa cells for 48 h. G stands for genistein. (d). Dose-dependent effects of genistein on untreated HeLa cell (upper figure) and ethanol-stressed HeLa cells (E8 hrs + G24 hrs, lower figure) for 24 h. Cont’ E: continuous ethanol stress treatment; E8 hrs + R24 hrs: ethanol stressed for 8 h and ethanol was replaced by fresh medium for 24 h recovery; Blank: DMSO solvent without genistein. (e). Annexin V and propidium iodide staining of HeLa cells before (untreated) and after stress treatment (E8 hrs), and after the removal of stress (E8 hrs + RXhrs). Groups treated with genistein (15 μg/ml) alone (G Alone) and the stressed groups treated with genistein (15 μg/ml) after stress removal (E8 hr + GXhrs) were included. Cont’ E for continuous ethanol stress treatment group. The percentage of events of early apoptotic (lower right), and late apoptotic or necrotic (upper right) is indicated in each diagram. (f). Morphological changes of HeLa cells after stress treatment (E8hrs), stress treatment and 24 h recovery (E8 hrs + R24 hrs), and recovery in presence of genistein (15 μg/ml) (E8 hrs + G24 hrs) under inverted phase contrast microscope (Scale bar: 10 μm). (g). Trypan blue dye exclusion assay of HeLa cells after stress treatment, stress treatment and 24 h recovery, and recovery in presence of genistein (15 μg/ml). (Data points in above figures represent Mean ± SEM of three independent experiments; SEM = error bar; * indicates: p < 0.05; ** indicates: p < 0.01).