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Figure 4 | BMC Cell Biology

Figure 4

From: Activation of α1A-adrenergic receptor promotes differentiation of rat-1 fibroblasts to a smooth muscle-like phenotype

Figure 4

Effect of PE and cAMP-elevating agents, FN and 8-cpt-cAMP, on DNA synthesis. Cells were serum-deprived for 2 days. A, Cells were treated with PE (5 μM), FN (0.1, 1, and 10 μM), and 8-cpt-cAMP (5, 10, and 20 μM) for 18 h. Incorporation of [3H]thymidine into rat-1 fibroblasts was determined as described in Methods. Data are expressed as dpm of [3H]thymidine incorporated per well. Values are the mean ± S.E. of three independent experiments performed in sextuplicates in different batches of cells. * Value significantly different from vehicle, p < .05. B, Effect of inhibitors of adenylyl cyclase inhibition on PE and cAMP-elevating agents. Cells were pre-incubated with SQ22536 (25 μM), an specific adenylyl cyclase inhibitor for 1 h. Cells were then treated with 5 μM PE and/or 1 μM FN for 18 h. [3H]thymidine incorporation was determined as described in Methods. Values are the mean ± S.E. of three independent experiments performed in quadruplicates on different batches of cells. Data are expressed as dpm of [3H]thymidine incorporated per well. * Value significantly different from the corresponding vehicle, p < .05. § value significantly different from that obtained in the presence of agonist alone, p < .05. C, Effect of transfection of πLXX-PKI [1-31] on PE-induced inhibition of DNA synthesis. Preconfluent cells were transiently transfected with πLXX-PKI [1-31], a plasmid encoding for the protein kinase inhibitor, (PKI), using Lipofectamine Plus transfection reagent according to the manufacturer's protocol (Life Technologies). Cells were cultured in serum-free DMEM for 24 h and then treated with 5 μM PE and/or 1 μM FN. Cells were assayed for [3H]thymidine incorporation 48 h post transfection as described in Methods. Data are expressed as dpm of [3H]thymidine incorporated per well. Values are the mean ± S.E. of three independent experiments performed on different batches of cells. * Value significantly different from the corresponding vehicle, p < .05. § value significantly different from that obtained in the presence of agonist alone, p < .05.

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