Figure 5.

Validation of SMIP001 and SMIP004. (a) Chemical structure of the two small molecule inhibitors of p27 depletions (SMIPs) identified in the screen. (b) Cell lysates from LNCaP-S14 treated with 40 μM SMIPs for 24 h were analysed by immunoblotting. Roscovitine (R) was used as positive control. The data shown is representative of two independent experiments. (c) LNCaP-S14 cells were treated with increasing concentrations of SMIPs (5-40 μM) for 18 h followed by fixation and staining for p27. The graph represents data from 24 replicate wells per concentration. (d) LNCaP-S14 cells were treated with SMIPs (5-40 μM) for 18 h and analysed by immunoblotting for the levels of p27 and p21. (e) p27, p21 and SKP2 stability in LNCaP-S14 cells treated with 40 μM SMIPs for 18 h. Cycloheximide (CHX, 100 μg/mL) was added for the indicated time points prior to preparation of cell lysates and immunoblot analysis. MG132 was used as positive control. The graphs represent the quantification of p27, p21 and SKP2 levels relative to the zero hour time point. The data is representative of three independent experiments. (f) LNCaP-S14 cells were treated with SMIP00s (40 μM) for 18 h followed by extraction of total RNA. The mRNA levels of p27, p21 and SKP2 were quantified by quantitative polymerase chain reaction as described in the Methods section. All data was normalized to GAPDH and is expressed as fold induction. The graph is representative of two independent experiments. (g) Total lysates of LNCaP-S14 cells treated with DMSO (-) or SMIPs (20 and 40 μM) for 24 h were analysed by immunoblotting using anti-ubiquitin antibody. The proteasome inhibitors, MG132 (20 μM) and bortezomib (100 nM) were used as positive controls. The blot is representative of two independent experiments.