Figure 6.

MKP1 overexpression in undifferentiated HESCs protects against ROS effects. A) Undifferentiated HESC cultures were either mock or MKP1-myc transfected (500 ng) and exposed to 250 μM H₂O₂ over a time course of 24 h. Whole-cell extracts were subjected to Western blot analysis with antibodies against SUMO-1, total- and phospho-JNK, myc tag, and β-actin. B) Undifferentiated HESC cultures were either mock or MKP1-myc transfected (500 ng) and exposed to 250 μM H₂O₂ for 8 h. RNA was extracted, and RTQ-PCR was used to determine the abundance of GADD45A, RRAD, and FOXO1 transcripts, relative to L19. Data are presented as fold induction relative to the transcript levels in untreated mock transfected cells and depict means ± sd triplicate determinations. Results are representative of 3 independent experiments. C) Primary HESCs were transfected with PR-A (400 ng) and PRE2-luciferase reporter (100 ng) with or without MKP1 expression vector (500 ng), and treated for 24 h with MPA and 0, 100, 250, and 500 μM H₂O₂, as indicated. Cells were harvested, and luciferase activity was assayed. Data are expressed as fold induction with regard to −MPA treatment and represent means ± sd of triplicate determinations.