Effects of celastrol on U937 culture proliferation and viability in vitro. Cells were seeded at 2 × 105/ml in 96-well culture plate and treated with indicated doses of celastrol for 1 d. A: Dose-dependent effects of celastrol on the number of total, living and dead cells. After treatment, the numbers of living and dead cells in each sample was determined by single-tube platform flow cytometry using self-made cell-Beads as an internal standard (detailed in Methods). Each data point represents the mean value of three test repetitions. B: Dot plot for flow cytometric analysis of living and apoptotic cells. The samples were labeled with Annexin V- FITC and PI double staining, and then detected by FCM. Living cells tested negative for both Annexin V and PI. Populations testing Annexin V positive/PI negative were in early-stage apoptosis, and double positives were in late-stage apoptosis. The percentages of each population are labeled in the corresponding plot region. C. Bar graph showing percentage of living, early, and late-stage apoptotic cells in samples with varying treatments. The values shown are the mean of three independent experiments.