Figure 6. ISO treatment increased mkp-1 mRNA stability.

(A) Cl41 cells were treated with medium or TPA/EGF (40 ng /ml) alone, or pretreated with 50μM of ISO for 30 min and then co-incubated with ISO and TPA/EGF for 12 hours. RT-PCR was performed to determine mkp-1 mRNA levels. The gapdh mRNA levels were used as loading control. (B) Cl41 cells stably transfected with MKP-1 promoter luciferase reporter were treated with medium or TPA/EGF (40 ng /ml) alone, or pretreated with 50 μM of ISO for 30 min followed by co-incubation with ISO and TPA/EGF for 24 hours. The luciferase activity was measured as described in “Materials and Methods”. The results were presented as relative MKP-1 promoter activity compared with that of the cells treated with TPA/EGF. The symbol (*) indicates a significant decrease as compared with cells treated with TPA/EGF alone (P<0.05). (C) Cl41 cells were pretreated with ISO (50 μM) for 6 hours and then co-incubated with ISO and actinomycin D (20 μM) for indicated time periods. Total RNA was isolated and RT-PCR was then performed to determine mkp-1 mRNA levels. The result was a representative one from three independent experiments. (D) The relative mRNA level of mkp-1 was determined by ImageQuant 5.2 (GE Healthcare). Natural logarithm of ratio [mkp-1]_t/[mkp-1]₀ was plotted against the time and the half life of mkp-1 mRNA was calculated via linear regression. (E) Cl41 cells were treated with ISO (50 μM) for indicated time periods. The total cell extracts were subjected to Western blot analysis with specific antibodies against HNRPD, VHL, Hur, Nucleolin, and MKP-1. (F) The diagram indicates mechanisms responsible for ISO inhibition of cell transformation.