I3C enhances TNF-induced cytotoxicity. (A) I3C enhances TNF- and chemotherapy-induced cytotoxicity. A total of 5000 Jurkat cells were seeded in triplicate in 96-well plates. Cells were pretreated with 1 μM I3C and then incubated with indicated concentrations of TNF (i), cisplatin (ii), or doxorubicin (iii) for 72 hours. Thereafter, cell viability was analyzed by the MTT method as described in “Materials and methods.” Values represent the mean ± SD of triplicate cultures. (B) Jurkat cells were pretreated with 25 μM I3C and then incubated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared, subjected to SDS-PAGE, and blotted with anti-PARP antibody. (C) Jurkat cells were pretreated with 25 μM I3C and then incubated with 1 nM TNF for 16 hours. Cells were stained with Live and Dead assay reagent for 30 minutes and then analyzed under a fluorescence microscope as described in “Materials and methods.” (D) Jurkat cells were pretreated with 25 μM I3C and then incubated with 1 nM TNF for 16 hours. Cells were fixed, stained with TUNEL assay reagent, and then analyzed under a fluorescence microscope as described in “Materials and methods.” Stained slides were mounted with mounting medium (Sigma-Aldrich) and analyzed under an epifluorescence microscope (Labophot-2; Nikon). Pictures were captured using a Photometrics Coolsnap CF color camera (Nikon, Lewisville, TX) and Meta Morph version 4.6.5 software (Universal Imaging). Original magnification, ×200.