Fig. 3

FKB downregulates Skp2 expression via protein degradation. a, Western blotting analysis of protein expression of Skp2 and p27/Kip1 in PC3 and C4-2B cells that were treated with vehicle control (0.1% DMSO) or indicated concentrations of FKB for 24 h. β-tubulin was used as a loading control. b, LNCaP cells were cultured under 10% charcoal stripped FBS with or without 10 nM synthetic androgen R1881 and then treated with vehicle control (0.1% DMSO) or 8.8 μM FKB for 24 h. The protein expression of Skp2 was decreased by FKB treatment. c, PC3 cells were treated with 0.5% DMSO, 8.8 μM FKB, 5 μM MG132 or 10 nM Bortezomib for 16 h. Real-time RT-PCR was performed to analyze mRNA expression of Skp2. PC3 cells were co-transfected with Skp2-Luc along with a Renilla luciferase plasmid pGL 4.71 and Luciferase activities were measured. Proteasome inhibitors MG132 and Bortezomib but not FKB significantly decrease mRNA expression and promoter activity of Skp2 (Student t test, Ps < 0.05). Bars are mean ± SD of three independent experiments. d, PC3 cells were treated with 10 μg/L of cycloheximide (CHX). After 16 h of treatment, the cells were treated in absence or presence of 8.8 μM of FKB. Western blotting was performed to determine Skp2 protein levels and quantified by densitometry with Image J software and adjusted for loading control. FKB reduces Skp2 protein stabilities over time