Figure 5

Inhibitory effect of p53 antisense upon forskolin-induced MMP-2 expression and secretion and trophoblast invasiveness. JAR cells were transfected with antisense against p53, then cultured in the presence or absence of 10 μM Forskolin, simultaneously with non-transfected cells. A,B, Representative photos taking with a confocal microscope of JAR cells, magnification × 60. JAR cells were fixed and stained with a fluoroscent antibody against p53 (A) or against MMP-2 (B), or nuclear proteins extracted and western blot performed (D). A-D Results represent 3 different experiments performed in duplicates. C, Density of p53 and MMP-2 staining was quantified with Image software and presented by Bar graph, black-p53, grey-MMP-2.D, representative pictures of western blot.E 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 semiquantative reverse transcription-PCR.E, 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 4 independent experiments performed in duplicates. F, MMP-2 secretion (72 kD) was assessed by zymography of conditioned media. F, Top panel: Representative zymography gels. F, Bottom panel, Bar graph, representing mean ± SEM from 3 independent experiments performed in quadruplets. The control band intensity was indicated as 100 percent. G, Bar graph representing cell invasion ability of transfected and non-transfected JAR cells tested with Transwell Invasion Assay from 3 different experiments performed in quadruplets.