Effect of ponicidin or oridonin on

Effect of ponicidin or oridonin on induction of apoptosis and cell cycle progression. Cell cycle phase distribution was analyzed by flow cytomFig. 1. Structural formulas of ponicidin and oridonin. etry. Cultures were incubated with 5 lM ponicidin or oridonin for 3 days and arvested. Cells were washed once with PBS and stained with 1.0 lg/ml 4,6-diamidion-2-phenylindole (DAPI; Molecular Probes, Eugene, OR) in a solution containing 100 mM NaCl, 2 mM MgCl2, and 0.1% Triton X-100, pH 6.8, as described [21,22]. The DNA-specific DAPI fluorescence was excited with UV light and collected with appropriate filters in an ICP-22 flow cytometer (Ortho Diagnostic,
Westwood, MA). The cell cycle distribution and percentage of apoptotic cells were analyzed by deconvoluting the DNA content frequency histograms with the use of CellFit software (Phoenix Flow, San Diego, CA), as detailed previously [22]. Protein extraction and Western blot analysis. Control and treated cells were rinsed with ice-cold PBS, suspended in buffer (50 ll/106 cells) containing 10 mM Hepes, pH 7.5, 90 mM KCl, 1.5 mM Mg(OAc)2, 1 mM dithiothreitol, 0.5% NP40, and 5% glycerol supplemented with 0.5 mM PMSF, 10 lg/ml each of aprotinin, pepstatin, and leupeptin, and lysed by three freeze/thaw cycles [18,19]. The extracts were centrifuged and the clear supernatants were stored in aliquots at
70 C. Protein concentrations were measured with protein assay reagent (Pierce Chem., Rockford, IL). For Western blot analysis, 10 lg proteins were boiled for 5 min in Laemmli buffer and separated by 10% SDS–polyacrylamide gel electrophoresis
(SDS–PAGE). The gels were transferred to nitrocellulose membranes by a semi-dry transfer method. After blocking with buffer containing 5% low-fat milk, the membranes were probed for the expression of cyclins B, D1,and E, cdc2, CDK 4, E2F, Rb, NF-jB p65 and p50, IjB, and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA). Site-specific Rb antibodies (Ser 780 and Thr 821) were purchased from Biosource International (Camarillo, CA). All antibodies used in the experiments were diluted at 1:1000. Specific immunoreactivity was demonstrated by
enhanced chemiluminescence (ECL) or color reaction using procedures detailed in the manufacturer
s protocol (Kirkegared & Perry Laboratories, Gaithersburg, MD).