Figure 2.

Prodigiosin up-regulates p27^KIP1 predominantly by increasing protein stability. A549, CL1-5 and H23 cells were treated with prodigiosin (0–100 nM) for 24 h. Total RNA was extracted thereafter and then subjected to (A) semi-quantitative RT-PCR and quantitative real-time RT-PCR analyses for mRNA expression of p21^CIP1 (B) and p27^KIP1 (C). Equal loading was confirmed by the levels of human actin mRNA. An evident increase in p21^CIP1 mRNA expression was observed, while the level of p27^KIP1 mRNA was barely changed. All experiments were repeated at least three times with triplicate samples in each experiment. Data are expressed as means ± SEM. *P < 0.05; **P < 0.01. (D) Prodigiosin shows limited effect on the CDKN1B promoter activity. A549, CL1-5 and H23 cells were transiently transfected with the p27^KIP1 promoter reporter plasmid pCDKN1B-Luc 24 h before prodigiosin treatment (100 nM). The extent of the p27^KIP1 promoter activity was assessed 24 h later by determining luciferase activity. (E) Prodigiosin stabilizes p27^KIP1. A549, CL1-5 and H23 cells were treated without or with prodigiosin (100 nM) in the presence of cycloheximide (60 µg·mL⁻¹) added 18 h after prodigiosin treatment. The protein levels of p27^KIP1 at 0, 1, 3 and 6 h following addition of cycloheximide were determined by immunoblotting analysis. Human β-tubulin was used as the loading control. (F) Prodigiosin extends the half-life of p27^KIP1 protein. ImageJ software was used to determine the intensity of p27^KIP1 protein signals on three independent immunoblots from cycloheximide experiments shown in (E). Upper panel: A549; middle panel: H23; lower panel: CL1-5. CHX, cycloheximide.