Mechanism of silvestrol-mediated Mcl-1reduction. (A) Effect of silvestrol on Mcl-1 transcription: RNA was extracted from CLL patient cells treated either with 80 nM silvestrol, 200 nM flavopiridol, or 100 μM cycloheximide (CHX) for 4 or 12 hours. n = 6 for untreated and silvestrol treated; n = 3 for flavopiridol and CHX treated. Mcl-1 message level was analyzed by real-time RT-PCR and was normalized relative to 18S RNA. Data are expressed as fold change in Mcl-1 message level over the time-matched untreated sample. Increases in Mcl-1 mRNA with silvestrol treatment were not significant. Bars show plus or minus SD. (B) Translation inhibition by silvestrol: In vitro translation reactions using in vitro–transcribed Mcl-1 mRNA were prepared in the presence or absence of silvestrol, cycloheximide, or flavopiridol as indicated. Reactions were separated by SDS-PAGE and detected using anti–Mcl-1 antibody. Translation assay control reactions contained luciferase mRNA or no mRNA. Lysate from the 697 cell line was included as an Mcl-1 protein control. This figure is from a single immunoblot; a vertical line was inserted to indicate the deletion of an irrelevant lane. (C) Silvestrol does not affect eIF2α phosphorylation: 697 ALL cells were treated with or without silvestrol (80 nM) or arsenic (200 μM) for 2.5 hours and lysates analyzed by immunoblot. HeLa cells were included as control. (D) Silvestrol does not affect 4EBP phosphorylation: 697 ALL cells were serum starved for 3 hours, then were treated with or without silvestrol (80 nM) or rapamycin (20 nM) for 2.5 hours in the presence of 20% fetal bovine serum. Lysates were analyzed by immunoblot.