Gossypin potentiates the apoptotic effects of TNF and chemotherapeutic agents. (A,B) Live/dead assay results indicate that gossypin upregulated TNF-induced cytotoxicity. KBM-5 cells were preincubated with 50 μM gossypin for 2 hours and then treated with 1 nmol/L TNF for 24 hours. Cells were stained with live/dead assay reagent for 30 minutes and then analyzed with a fluorescence microscope, as described in “Materials and methods.” (C) Gossypin enhanced Taxol-induced cytotoxicity. KBM-5 cells were pretreated with 50 μM gossypin and then incubated with 3 nmol/L of Taxol for 24 hours. Thereafter, cell viability was analyzed by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide method, as described in Materials and methods. (D) Cells were pretreated with 50 μM gossypin for 2 hours and then incubated with 1 nM TNF for 16 hours. Cells were incubated with anti-annexin V Ab conjugated with FITC and then analyzed with a flow cytometer to identify early apoptotic effects. (E) TUNEL staining shows that TNF-induced apoptosis was enhanced by incubation with gossypin. KBM-5 cells were preincubated with 50 μM gossypin for 2 hours and then treated with 1 nmol/L TNF for 16 hours. Cells were fixed, stained with TUNEL assay reagent, and analyzed by flow cytometry, as described in Materials and methods. (F) Gossypin potentiated TNF-induced apoptosis. KBM-5 cells were preincubated with 50 μM gossypin for 2 hours and then treated with 1 nmol/L TNF for the indicated times. Whole-cell extracts were prepared, subjected to SDS-PAGE, and blotted with anti-PARP antibody.