Effects of IL-2 supplementation on cell growth, apoptosis, and ceramide content and inhibition of IL-2-induced cell growth by exogenous C2-ceramide in KHYG-1 cells. (A) KHYG-1 cells were cultured in the presence (•, IL-2⁺) or absence (○, IL-2^-) of IL-2 (100 U/mL) for 36 hours. After IL-2 deprivation for 12 hours, cells were rescued with IL-2 (100 U/mL) supplementation and further cultured for 24 hours (⋄, IL-2^-→+). Cells were harvested at the indicated times (0, 12, 24, and 36 hours). Viable cell numbers and the percentage of apoptotic cells (inset) were counted by a trypan blue dye exclusion method and DAPI method under fluorescent microscope, respectively. (B) Intracellular ceramide was measured by DGK assay after lipid extraction by Bligh and Dyer method as described in “Materials and methods.” (C-D) KHYG-1 cells at an initial concentration of 3 × 10⁵ cells/mL were treated with various concentrations of C2-ceramide (0, 20, 35, and 50 μM) in the presence or absence of IL-2 for 24 hours. Viable cell number and ceramide content were assessed by trypan blue dye exclusion method and DGK assay, respectively. The results were obtained from 3 different experiments. The bars indicate 1 SD. The significance of difference of cell count was determined by analysis of variance (ANOVA) test. ^*P < .01.