Effect of chrysin on proteins that regulate cell cycle. (A) The effect of chrysin on Cdk4, Cdk2, Cyclin D1, p53, p21 and p27 proteins were determined by Western blot analysis from the total protein extracted from A375 cells after incubating with 40 μM of Chrysin and 4 μM of TSA for a time period of 24 h. The blots were reprobed with Tubulin antibody that acts as gel loading control. C is the control untreated cells. C + D: is the control cells treated with DMSO (0.1 %). Bar diagram representing the intensity of expression of p21, p53, p27, cyclin D1, Cdk2 proteins. (B) The induction of p21 protein by compounds Chrysin 40 μM(Chry) and TSA 4 μM was studied by western blot analysis in K562 leukemia cells which is null for p53 and U3A, fibrosarcoma cells which is null for STAT-1 . We observed induction of p21 was independent of p53 and dependent on STAT-1. C: Control, untreated cells and C + D represents control untreated cells incubated with 0.1 % DMSO. Each experiment was conducted three times and standard deviations were derived. Data represents mean ± S.D of three independent experiments performed. *** represents P < 0.001; ** represents p < 0.01. (C) The effect of chrysin on mRNAlevels of p21 in K562 (p53−/−) and U3A (STAT1 −/−) cells. P21 induction was observed 3–4 folds in chrysin (40 μM) treated K562 cells but not in U3A cells which lack STAT-1 . These treatments were carried out for 24 h (D) Transcriptional run-on assays using radio-labeled nuclear RNA extracted from 0.1 % DMSO and chrysin (40 μM) treated A375 cells after 24 hrs. Actin nuclear RNA was used as internal control. The relative ratio of P21/ Actin from triplicate blots were shown in a bar chart. *** represents P < 0.001; ** represents p < 0.01. (E) The quantitative real-time PCR assay for the p21 mRNA in chrysin (40 μM) and TSA (4 μM) treated A375 cells for 24 h. Control indicates cells treated with DMSO (0.1 %).