In experiment 1, during the 24-h serum withdrawal period, 100 nM PMA was added at 0, 6, or 24 h before collection of culture medium and recovery of the cells. In experiment 2, 72 h before expected confluency, the culture medium was replaced by fresh RPMI supplemented with 10% dextran-coated charcoal-treated fetal calf serum. During this period, 4 sex steroid treatment groups were analyzed. In treatment group 1, cells were treated with es-tradiol-17p (E2, 1 nM) for 72 h. buy cheap antibiotics
In treatment group 2, cells were treated with ethanol (0.1%) for 48 h, then with progesterone (P4, 10 nM) the last 24 h. In treatment group 3, cells were treated with dexamethasone (DEX) 24 h after a 48 h pretreatment with ethanol (0.1%). In treatment group 4, cells were treated with E2 (1 nM) for 72 h plus P4 (10 nM) the last 24 h. The control group corresponded to cells treated with 0.1% ethanol (vehicle) alone (72 h). In all cases, 3 h (corresponding to the maximal mRNA expression induced by PMA, data not shown) or 6 h (maximal protein expression) before the end of the incubation, PMA (100 nM) was added to the cultures.