Silvestrol effects on Mcl-1 expression. (A) Silvestrol mediates caspase-independent Mcl-1 reduction: CLL cells treated with 80 nM silvestrol, 100 μM cycloheximide (CHX), or 25 μM F-ara A for 16 hours with or without caspase inhibitors Z-VAD-fmk or Boc-D-fmk (100 μM) were analyzed for Mcl-1 protein expression by immunoblot. Results are shown from 1 of 4 identical experiments. All samples tested to date showed substantial Mcl-1 protein reduction by this time point after silvestrol treatment. (B) Silvestrol-mediated Mcl-1 reduction in 697 acute lymphoblastic leukemia (ALL) cells is distinct from caspase activity: 697 ALL cell line was incubated for 8 hours with or without 80 nM silvestrol or 200 nM flavopiridol, and lysates were examined by immunoblot. (C) Silvestrol-mediated Mcl-1 reduction is independent of proteasome activity: CLL patient cells were treated with 80 nM silvestrol, 100 μM cycloheximide (CHX), or 200 nM flavopiridol for 4 or 8 hours, with or without 50 nM bortezomib. Lysates were analyzed for PARP and Mcl-1 expression by immunoblot.