FIG. 2 .
2Gap junctional coupling in osteoblastic cells. Functional coupling was reduced more than 9-fold in confluent cultures of antisense transfectants RCx4 (1.2 Ϯ 1.1) and RCx16 (1.5 Ϯ 1.3) and in subconfluent ROS (ROS-s; 2.4 Ϯ 1.4) and subconfluent bG (bG-s; 2.1 Ϯ 1.6) as compared with confluent ROS or bG (15.5 Ϯ 2.4 and 13.9 Ϯ 1.9, respectively). Each bar represents the mean (ϮS.E.) number of adjacent cells that take up dye within 5 min. *, significantly different from either ROS or bG (p Ͻ 0.01; n ϭ 25-50 cells). FIG. 3. Effect of dbcAMP on gap junctional coupling in osteoblastic cells. Cells plated at 50,000 cells/cm 2 were exposed to either 10 M dbcAMP or vehicle control. Functional coupling was quantified by dye injections with 0.1 mM carboxyfluorescein at room temperature. The number of neighboring cells with dye was recorded every 5 min for 80 min. The addition of dbcAMP increased coupling in ROS (thick solid line) and bG (thick dashed lines) by 50% within 20 or 30 min, respectively. The inability of antisense transfectants RCx4 (thin solid line) and RCx16 (thin dashed line) to significantly up-regulate functional coupling in response to dbcAMP demonstrates that Cx43 expression had been successfully reduced and that neither recruitable membrane nor cytoplasmic Cx43 protein reserves were sufficiently available to enable maximal dye coupling. Each **data** point represents the mean (ϮS.E.) number of cells coupled (n ϭ 3). *, significantly different from basal values within each group (p Ͻ 0.01). FIG. 4. Connexin43 and GAPDH mRNA expression in osteoblastic cells. 20 g of total RNA was subjected to Northern blot analysis using either a 890-base pair rat Cx43 cDNA radiolabeled probe (A) or a 1.4-kb rat GAPDH radiolabeled probe (B). RCx4 clones displayed a lack of Cx43 mRNA (A, lane 3), whereas RCx16 displayed a decreased expression (A, lane 4) relative to ROS (A, lane 1) and bG (A, lane 2). All cells examined displayed equivalent mRNA expression of the housekeeping gene, GAPDH (B). These **data** are typical of three similar experiments.
