Fig. 7. Attenuation of CX43 expression in human endometrial stromal cells leads to impaired gap junction communication, decidualization and a reduction in VEGF production. (A) Western blotting was used to analyze the expression of CX43 in HESC-T cells stably transfected with a CX43 siRNA (HESC-T3) or a nonsilencing control vector (HESC-TC). Pronounced silencing of CX43 protein was observed in HESC-T3 cells, whereas the levels of beta actin protein remained unaltered. (B) Gap junction intercellular communication was determined using a double-labeling technique. Donor cells were double labeled with the fluorescent dyes calcein (gap junction permeable dye, green fluorescence) and DiI (gap junction impermeable dye, red fluorescence), and were then placed in contact with unloaded cells in the monolayer. Dye transfer was visualized after 2 hours. HESC-T3 cells with reduced CX43 expression exhibited lack of green dye diffusion. Arrows show examples of functional coupling between HESC-TC cells. (C) HESC-TC and HESC-T3 cells were grown in DMEM/F-12 medium containing 5% charcoal-stripped FBS. To induce in vitro decidualization, they were treated with or without a hormone cocktail containing 1 nM E, 1 mM P and 0.5 mM 8-bromo-cAMP for 7-11 days (Ryan et al., 1994). When the cells were examined morphologically, a distinct transition from fibroblastic to a plump, epitheloid phenotype, characteristic of decidual cells, was observed in control HESC-TC cells. Low CX43-expressing HESC-T3 cells failed to show this morphological transformation. (D) HESC-TC and HESC-T3 cells were grown for 24 hours in the absence (Con) or presence (TPA) of 50 nM phorbol ester. The VEGF in supernatant was measured in duplicate by ELISA.