Effects of pristimerin on cyclin D1, D2, and D3 protein expression, cell-cycle progression, and viability of human myeloma cell lines. (A) Immunoblot showing suppression of cyclins D1, D2, and D3 by pristimerin (500 nM) at 14 hours in H929, KMS11, and U266 human myeloma cells. Pristimerin effects are compared with DMSO vehicle. (B) Temporal course of cyclin D1, D2, and D3 suppression in H929 following pristimerin (500 nM) exposure, showing significant suppression by 6 hours, increasing to 16 hours. (C) Cell-cycle profiles of H929 and U266 cells grown in standard media and treated with vehicle or pristimerin (500 nM) at 16 hours. Major left and right peaks represent G₀/G₁ and G₂/M phases, respectively. Pristimerin causes expansion of a sub-G0 fraction, corresponding to apoptotic cells. Similar results were obtained with duplicate experiments. (D) Inhibition of heterogeneous myeloma cell lines by titrated pristimerin, assessed by MTT assay at 72 hours. Results at each concentration represent the mean of triplicate wells ± SEM. At the left of the dashed line, the background proliferative activity of each cell line is presented, with MTT results shown for cells at day 0 and for untreated cells at assay completion (0 nM). The median IC₅₀ of pristimerin against myeloma cell lines is approximately 200 nM. (E) Myeloma growth cytokines IL-6 (10 ng/mL), IGF-1 (100 ng/mL), and Baff (25 ng/mL) fail to rescue cells from pristimerin. Viability was assessed by MTT assay at 72 hours. Error bars represent the standard deviation of triplicate assay results.