Figure 4

Inhibitory effect of Ets-2 antisense upon forskolin-induced MMP-2 expression and secretion and trophoblast invasiveness. A, JAR cells were transfected with antisense against Ets-2 (As Ets-2), then cultured in the presence or absence of 10 μM Forskolin, simultaneously with non-transfected cells, then fixed and stained with a fluorescent antibody against Ets-2 (A), or nuclear proteins extracted and western blot performed (C). The results are representative photos taking with a confocal microscope, magnification × 60. A-C Results represent 3 different experiments performed in duplicates. B, Density of ETS-2 staining was quantified with Image software and presented by Bar graph. C, representative pictures of western blot. D, Top panel, Total RNA of MMP-2 from JAR cell, with or without treatment with Ets-2 antisense and in the presence or absence of Forskolin was assessed by semi-quantitative reverse transcription-PCR. D, Bottom panel, band intensities were quantified with Densitometer system and presented as ratio of MMP-2 to GAPDH, as percent of control. Data represents mean ± SEM from 3 independent experiments performed in quadruples. E, MMP-2 secretion (72 kD) was assessed by zymography of conditioned media. E Top panel: Representative zymography gels. E, Bottom panel, Bar graph, representing mean ± SEM from 3 independent experiments performed in quadruplets. The control band intensity was indicated as 100 percent. F, Bar graph representing cell invasion ability of transfected and non-transfected JAR cells tested with Transwell Invasion Assay from 4 different experiments performed in duplicates.