Figure 3.

Dose-dependent inhibition of basal and GnRH-induced MAPK1/3 activation by OLA. LβT2 cells were serum starved for 12 to 16 hours, treated with vehicle or OLA for 3 hours, and then stimulated with 10 nM GnRH for the final 15 minutes. Cells were harvested after incubation and protein extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot for phosphorylated and total MAPK1/3. (A) Representative chemiluminescent image of phosphorylated (p-) and total (t-)MAPK1/3. (B) Summary of six independent experiments of OLA- and GnRH-induced MAPK1/3 phosphorylation reported as fold change from vehicle-treated controls. Bars represent the ratio of p-MAPK1/3 to t-MAPK1/3 normalized to vehicle control. *Significant difference from positive control GnRH treatment alone as determined by ANOVA and Dunnett post hoc test; ^#significant difference from normalized vehicle control as determined by Student t test. Results indicate that OLA reduces basal activation of MAPK1/3 and abolishes the stimulatory effect of GnRH on MAPK1/3 phosphorylation at the 1000-μM dose. To assess the dependence of activation on ROS, LβT2 cells were treated as in A with vehicle or 500 μM OLA for 3 hours with the addition of vehicle or 1 mM NAC 30 minutes prior to the addition of OLA. Cells were subsequently treated with vehicle or 10 nM GnRH in the final 15 minutes and evaluated after harvest for MAPK1/3 activation as above. (C) Representative chemiluminescent image of p- and t-MAPK1/3. (D) Summary of six independent experiments showing NAC-sensitive activation of MAPK1/3 by GnRH and OLA. Bars represent the ratio of p- to total MAPK1/3. Error bars denote standard error of the mean of at least three independent experiments. *Significant difference from vehicle control as determined by ANOVA and Dunnett post hoc test; ^#significant difference of NAC-treated group from the corresponding untreated group as determined by Student t test. w/o, without.