Mouse dermal fibroblasts express Crh and its receptors.

<p>(a) Total RNA isolated from whole brain from <i>Crh+/+</i> mice (lane 1), NMF isolated from <i>Crh+/+</i> mice (lane 2) and NMF isolated from <i>Crh−/−</i> mice (lane 3) was subjected to RT-PCR for evaluation of <i>Crh</i> expression. Lane 4 represents a sample without RT-enzyme and lane 5 represents a sample without template. (b) <i>Crf1</i> mRNA expression in NMF isolated from <i>Crh+/+</i> mice (lane 2). Lane 1 represents <i>Crf1</i> mRNA expression isolated from whole brain, while lane 3 represents a sample without RT-enzyme and lane 4 a sample without template. (c) <i>Crf2</i> mRNA expression in NMF isolated from <i>Crh+/+</i> mice (lane 2). Lane 1 represents <i>Crf1</i> mRNA expression isolated from heart, while lane 3 represents a sample without RT-enzyme and lane 4 a sample without template. (d) Effects of antalarmin and astressin-2B on specific [¹²⁵I] Tyr0-sauvagine binding to CRF₁ and CRF₂. Membrane homogenates from <i>Crh+/+</i> and <i>Crh−/−</i> fibroblasts were assayed for specific binding with [¹²⁵I] Tyr0-sauvagine, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021654#s4" target="_blank">Materials and Methods</a>. The bars represent the % decrease of specific binding. The mean ± SEM values are from 3 independent experiments, each performed with duplicate determinations. (e) Effect of CRH on cAMP accumulation in <i>Crh+/+</i> and <i>Crh−/−</i> fibroblasts. Stimulation of cAMP accumulation by CRH was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021654#s4" target="_blank">Materials and Methods</a> in intact cells. The mean ± SEM values are from 3 independent experiments, each performed with duplicate determinations. * represents statistical difference (P<0.05) between genotypes exposed to the same treatment and # represents statistical difference (P<0.05) between different treatments in the same genotype.</p

Enhanced migratory response of Crh−/− fibroblasts.

<p>Fibroblasts isolated from <i>Crh+/+</i> and <i>Crh−/−</i> dermis were allowed to attach on the dish. A clear space was produced in the confluent monolayer, cultured in serum free or serum treated with 10 µg/ml mytomycin and the wounded fibroblast layer was photographed immediately and 24 and 48 h after wounding. The distance of migration from the original borders was determined and is indicated (b) as a percentage of the original distance in a representative experiment. * represents statistical difference (P<0.05) between genotypes. (n = 3 wells/treatment/experiment, at least 4 independent experiments).</p

IL-6 and TGF-β1 production from Crh+/+ and Crh−/− fibroblasts.

<p>Primary dermal fibroblasts isolated from <i>Crh+/+</i> and <i>Crh−/−</i> mice were cultured in 24-well plates (10⁵ cells/well). Supernatants were collected and analyzed by ELISA for IL-6 (a) or TGF-β1 (b). * represents statistical difference (P<0.05) between genotypes (n = 3 wells/condition/experiment, at least 3 independent experiments).</p

Proliferative response of Crh+/+ and Crh−/− fibroblasts.

<p>(a) Fibroblasts isolated from <i>Crh+/+</i> and <i>Crh−/−</i> dermis plated at an initial density of 8×10³ cells/well for four days. * represents statistical difference (P<0.05) between genotypes. (n = 4 wells/treatment/experiment, at least 5 independent experiments). (b) Fibroblasts isolated from both genotypes cultured at an initial density of 10⁵ cells/well in serum supplemented or serum free medium for 30 hr and apoptosis was measured by staining cells with Annexin V/Propidium Iodide and flow cytometry. The experiment was performed three times, with representative results of one experiment shown here.</p

Effect of the CRH antagonists in human dermal fibroblasts.

<p>(a) HDFs isolated from human foreskin were cultured in 96-well plates (8,000 cells/well) in the presence of 100 nM antalarmin or ethanol (control). Proliferation was measured using either MTT or thymidine incorporation. *: represents statistical difference (P<0.05) between treatments (n = 4 wells/treatment/experiment, at least 3 independent experiments). (b) HDFs were allowed to attach on the 100 mm dish. A clear space was produced in the confluent monolayer, cultured in serum free or serum treated with 10 µg/ml mytomycin and the wounded fibroblast layer was photographed immediately and 24 h after wounding (n = 3 wells/treatment/experiment, at least 3 independent experiments). (c) HDFs were cultured in 24-well plates (10⁵ cells/well) in the presence of 100 nM antalarmin or ethanol (control). Supernatants were collected and analyzed by ELISA. * represents statistical difference (P<0.05) between treatments (n = 3 wells/condition/experiment, at least 3 independent experiments). (d) HDFs were cultured in 96-well plates (8,000 cells/well) in the presence of 1000 nM astressin 2B or ethanol (control). Proliferation was measured with MTT, (n = 4 wells/treatment/experiment, at least 3 independent experiments).</p