Validation of prioritized small molecule inhibitors of p27 depletions (SMIPs). (a) Accumulation of p27 upon treatment with SMIPs. LNCaP-S14 cells were treated for 18 h with the respective SMIPs (40 μM), DMSO (-) or the positive control MG132 (M). The data is representative of three independent experiments. (b) Quantification of three independent immunoblotting experiments by film densitometry and normalization to the loading control tubulin. (c) Target pathway selectivity was evaluated in LNCaP cells stably transfected with NKX3.1-YFP. Cells were treated with 40 μM SMIPs for 18 h. YFP fluorescence was normalized to cell numbers and plotted relative to DMSO treated cells. The graph represents the mean +/- standard deviation (SD) from 16 replicates. (d) Cell type specificity was evaluated by measuring the accumulation of p27Luc in Hela_p27Luc cells. Luciferase activity was measured in total cell lysate from cells treated with 40 μM of the respective SMIPs. The graph is representative of three independent experiments and shows the mean +/- SD from eight replicate measurements. Proteasome inhibitors MG132 and epoxomycin were used as positive controls.