Figure 1.
(A) The effect of DETA-NONOate on MDA-MB-231 viable cell count. Cells (7.5 × 105) were treated with 1 mM DETA-NONOate. The medium was changed and fresh drug was added daily. Viability of the cells was assessed every 12 h, up to 48 h, by trypan blue exclusion. The results are representative of four separate experiments (±SE). (B) FACS analysis of 1 mM DETA-NONOate-treated MDA-MB-231 cells at various time points. The cells were prepared for FACS analysis, as described in Materials and Methods, and analyzed in a FACScalibur flow cytometer. The number of cells in each phase of the cell cycle was obtained by modfit software. (C) Effect of DETA-NONOate (1 mM) on cell cycle proteins in MDA-MB-231 cells at different time points. Cells were harvested and prepared for Western blot analysis for cyclin D1, cyclin E, CDK4, CDK6, and CDK2 as described in Materials and Methods. (D) Densitometric scan of Western blot of cyclin D1 from C. Only the arbitrary densitometric units of 0, 24, and 48 h are plotted. (E) Effect of DETA-NONOate on phosphorylation status of pRb in MDA-MB-231 cells at different time points. Western blot analysis shows the phosphorylation status of pRb in the presence of DETA-NONOate. Hypophosphorylated pRb migrates faster than hyperphosphorylated pRb. (F) Western blot analysis showing reversibility of the effect of DETA-NONOate on cyclin D1. MDA-MB-231 cells were treated with DETA-NONOate (1 mM) for either 24 or 48 h. After treatment, the cells were washed and kept in DETA-NONOate-free medium for another 24 h, harvested, and prepared for immunoblotting with cyclin D1 antibody. Lane 1, control cells at 24 h; lane 2, DETA-NONOate treatment for 24 h; lane 3, cells treated with DETA-NONOate for 24 h were washed and allowed to recover for 24 h; lane 4, control cells for 48 h; lane 5, DETA-NONOate treatment for 48 h; lane 6, cells treated with DETA-NONOate for 48 h were washed and allowed to recover for 24 h.