Fig. 7. Effect of inhibition of phosphatase activity using okadaic acid on phospho-JNK levels.

HeLa cells were pre-treated with okadaic acid (100 nM) or the carrier control for 2 hr prior to UV irradiation (100 mJ cm⁻²). Cells were returned to either prewarmed treatment media or carrier control containing media immediately following irradiation and the kinetics of PJNK accumulation determined. (A,B) Treatment of uninfected cells with okadaic acid failed to increase the levels of phospho-JNK relative to the carrier control. Infected cells treated with both carrier control and okadaic acid exhibit lower levels of phospho-JNK compared to treatment and time matched uninfected cells. However, comparison of phospho-JNK levels between control and okadaic acid treated cells suggest a potential increase in relative phospho-JNK levels. (C,D) Densitometric analysis compiling the results from three independent experiments confirm the absence of any effect from okadaic acid in uninfected cells except possible a marginally increased loss of signal in okadiac acid treated cells. In contrast, treatment of infected cells with okadaic acid results in a significant increase in the levels of phospho-JNK relative to total JNK which are comparable to those seen in uninfected cells. This suggests an okadaic acid sensitive activity is partially responsible for the reduced steady state levels of phospho-JNK following UV stimulation. 2-way ANOVA analysis revealed highly significant effect for time post UV (p<0.0001). Okadaic acid treatment exhibited no significant effect in uninfected cells but was moderately significant in infected cells (p=0.0265). Error bars represent standard error of the mean from three independent experiments.