Fig. 1. Effects of Cx43 antisense on Cx43 protein production in human trophoblasts. (A) Oligonucleotide uptake by primary cytotrophoblasts. After 4 hours of culture, cytotrophoblasts were incubated with FITC-labeled scrambled oligonucleotide for 1 hours, 2 hours, 24 hours and 48 hours. At each time-point, cells were washed three times in PBS, fixed and analyzed by fluorescence microscopy. Nuclei were stained in blue by DAPI. Intensely fluorescent green cells have internalized the scrambled antisense oligonucleotide (FITC) (x600). (B) Immunodetection of Cx43 in cytotrophoblast cells isolated from normal placentas. Cells were treated for 48 hours with a scrambled (control) or a specific Cx43 antisense (Cx43 antisense). Cell nuclei were labeled with DAPI (blue immunofluorescence). In the control, Cx43 punctuate immunofluorescence (IF) can be observed around nuclei and at the borders with neighboring trophoblastic cells. In cells treated with Cx43 antisense, the level of IF is largely decreased (x1000). (C) Cx43 protein levels in trophoblast cells after treatment with a scrambled antisense (control) or with a specific Cx43 antisense (Cx43 AS) were determined by western blotting using a mouse anti-Cx43 monoclonal antibody. An anti-actin monoclonal antibody was used as a standard. Proteins obtained from rat brain lysate were used as a positive control. One representative experiment out of the three performed is shown.