Journal: Respiratory Research

Article Title: Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27 kip1

doi: 10.1186/1465-9921-8-77

Figure Lengend Snippet: Effects of hypoxia and BPS on BrdU incorporation in cultured HPASMC . Cultured HPASMC were exposed to various concentrations of oxygen and BPS in the presence of BrdU for 24 hours. (a) Severe hypoxia (0.1% oxygen) suppressed, whereas moderate hypoxia (2% oxygen) significantly enhanced BrdU incorporation. *P < 0.05 versus 21% oxygen. (b) BrdU incorporation was dose-dependently suppressed by BPS under both normoxic and hypoxic conditions. Data are expressed as means ± SE (n = 6). Open bars, 21% oxygen; solid bars, 2% oxygen. *P < 0.05 versus 21% oxygen control; † P < 0.05 versus without BPS.

Article Snippet: HPASMC supplied by Kurabo Ltd. (Osaka, Japan) were cultured in Humedia SG medium containing 5% fetal bovine serum, with 50 μg/ml of gentamycin, 50 ng/ml of amphotericin B, 1 ng/ml of recombinant human EGF, and 1 ng/ml of recombinant human FGF.

Techniques: BrdU Incorporation Assay, Cell Culture, Control

Journal: Respiratory Research

Article Title: Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27 kip1

doi: 10.1186/1465-9921-8-77

Figure Lengend Snippet: Cell cycle analysis of HPASMC exposed to hypoxia and BPS . Cultured HPASMC were exposed to 21% and 2% oxygen with or without 10 μM BPS for 24 hours. Cells were harvested and DNA fragmentation was analyzed using flow cytometry and propidium iodide staining. Area definitions on DNA histograms: C, G 0/1 phase; D, S phase; E, G 2 /M phase. Moderate hypoxia (2% oxygen) increased, whereas BPS significantly decreased ratios of S plus G 2 /M and G 2 /M phases. Histograms are representative and bar graph shows data expressed as means ± SE (n = 4). Open bars, ratios of G 2 /M phases; solid bars, ratios of S plus G 2 /M phases. *P < 0.05 versus 21% oxygen control; † P < 0.05 versus 2% oxygen without BPS.

Article Snippet: HPASMC supplied by Kurabo Ltd. (Osaka, Japan) were cultured in Humedia SG medium containing 5% fetal bovine serum, with 50 μg/ml of gentamycin, 50 ng/ml of amphotericin B, 1 ng/ml of recombinant human EGF, and 1 ng/ml of recombinant human FGF.

Techniques: Cell Cycle Assay, Cell Culture, Flow Cytometry, Staining, Control

Journal: Respiratory Research

Article Title: Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27 kip1

doi: 10.1186/1465-9921-8-77

Figure Lengend Snippet: Effects of BPS on intracellular cAMP production during hypoxia . Cultured HPASMC were exposed to 21% or 2% oxygen in the presence or absence of 10 μM of BPS for indicated periods. Concentrations of cAMP in cell lysates were measured using low-pH cAMP ELISA kits. Although BPS significantly induced cAMP expression, intracellular cAMP expression did not significantly differ between the indicated oxygen concentrations. Line with solid circles, 21% oxygen; dotted line with open circles, 2% oxygen. Data are expressed as means ± SE (n = 6).

Article Snippet: HPASMC supplied by Kurabo Ltd. (Osaka, Japan) were cultured in Humedia SG medium containing 5% fetal bovine serum, with 50 μg/ml of gentamycin, 50 ng/ml of amphotericin B, 1 ng/ml of recombinant human EGF, and 1 ng/ml of recombinant human FGF.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Respiratory Research

Article Title: Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27 kip1

doi: 10.1186/1465-9921-8-77

Figure Lengend Snippet: Effects of BPS and 8-Br-cAMPon p27 kip1 mRNA expression during hypoxia . Cultured HPASMC were exposed to 21% or 2% oxygen concentrations with or without 10 μM of BPS or 1 mM 8-Br-cAMP for indicated periods. Expression of p27 kip1 mRNA was measured using Real-time RT-PCR using LightCycler™. (a) BPS suppressed p27 kip1 mRNA reduction under both normoxic and hypoxic conditions. Expression of p27 kip1 mRNA between normoxic and hypoxic conditions did not significantly change. Graph shows ratio of p27 kip1 to β-actin mRNA expression. Open and solid bars, 21% and 2% oxygen, respectively. Data are expressed as means ± SE (n = 6). *P < 0.05 versus control. (b) Agarose gel electrophoresis with EB staining revealed single amplification of predicted PCR products (lane 1, DNA molecular weight markers; lane 2, p27 kip1 ; 109 bp; lane 3, β-actin 144 bp).

Article Snippet: HPASMC supplied by Kurabo Ltd. (Osaka, Japan) were cultured in Humedia SG medium containing 5% fetal bovine serum, with 50 μg/ml of gentamycin, 50 ng/ml of amphotericin B, 1 ng/ml of recombinant human EGF, and 1 ng/ml of recombinant human FGF.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Control, Agarose Gel Electrophoresis, Staining, Amplification, Molecular Weight

Journal: Respiratory Research

Article Title: Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27 kip1

doi: 10.1186/1465-9921-8-77

Figure Lengend Snippet: Effect of BPS and 8-Br-cAMP on p27 kip1 mRNA stability during hypoxia . Cultured HPASMC were exposed to 21% or 2% oxygen concentrations with or without 10 μM of BPS or 1 mM 8-Br-cAMP for indicated periods. The p27 kip1 mRNA stability was measured after adding 400 nM of Act D using Real-time RT-PCR using LightCycler™. Degradation of p27 kip1 mRNA was significantly suppressed by BPS and 8-Br-cAMP under both normoxic and moderately hypoxic conditions, and mRNA stability was slightly decreased by moderate hypoxia. Graphs show % maximal p27 kip1 mRNA expression. Line with solid circles, 21% oxygen; dotted line with open circles, 2% oxygen; line with solid squares, 21% oxygen and BPS; line with solid triangles, 21% oxygen and 8-Br-cAMP; dotted line with open squares, 2% oxygen and BPS; dotted line with open triangle, 2% oxygen and 8-Br-cAMP. Data are expressed as means ± SE (n = 6). *P < 0.05 versus 21% oxygen. † P < 0.05 versus oxygen controls.

Article Snippet: HPASMC supplied by Kurabo Ltd. (Osaka, Japan) were cultured in Humedia SG medium containing 5% fetal bovine serum, with 50 μg/ml of gentamycin, 50 ng/ml of amphotericin B, 1 ng/ml of recombinant human EGF, and 1 ng/ml of recombinant human FGF.

Techniques: Cell Culture, Quantitative RT-PCR, Expressing

Journal: Respiratory Research

Article Title: Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27 kip1

doi: 10.1186/1465-9921-8-77

Figure Lengend Snippet: Effects of BPS and 8-Br-cAMP on p27 kip1 protein expression during hypoxia . Cultured HPASMC were exposed to 21% and 2% oxygen with or without 1 mM of 8-Br-cAMP or 10 μM of BPS for 24 hours, and then Western blotted. Hypoxia decreased p27 kip1 protein expression. BPS and 8-Br-cAMP each significantly increased p27 kip1 protein expression under both normoxia and hypoxia. Photomicrographs are representative of 4 similar experiments, and bar graphs show density ratios of p27 kip1 protein versus those of β-actin bands. Open and solid bars, 21% and 2% oxygen, respectively. Data are expressed as means ± SE (n = 4). *P < 0.05 versus 21% oxygen control. † P < 0.05 versus 2% oxygen.

Article Snippet: HPASMC supplied by Kurabo Ltd. (Osaka, Japan) were cultured in Humedia SG medium containing 5% fetal bovine serum, with 50 μg/ml of gentamycin, 50 ng/ml of amphotericin B, 1 ng/ml of recombinant human EGF, and 1 ng/ml of recombinant human FGF.

Techniques: Expressing, Cell Culture, Western Blot, Control

Journal: Respiratory Research

Article Title: Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27 kip1

doi: 10.1186/1465-9921-8-77

Figure Lengend Snippet: Effects of BPS, 8-Br-cAMP, hypoxia, and PDGF on p27 kip1 protein stability . Cultured HPASMC were exposed to 21% or 2% oxygen with or without 10 μM BPS, 1 mM 8-Br-cAMP, or 25 ng/ml PDGF and 25 μg/ml of CHX for indicated periods and Western blotted. Degradation of p27 kip1 expression did not significantly change among cells exposed to hypoxia, BPS, or 8-Br-cAMP. PDGF promoted degradation of p27 kip1 protein expression. Graphs show % of maximal p27 kip1 protein expression. Line with solid circles, 21% oxygen (control); dotted line with open circles, 2% oxygen (hypoxia); line with solid squares, PDGF; dotted line with open triangles, BPS; dotted line with solid triangles, 8-Br-cAMP. Data are expressed as means ± SE (n = 4). *P < 0.05 versus 21% oxygen.

Article Snippet: HPASMC supplied by Kurabo Ltd. (Osaka, Japan) were cultured in Humedia SG medium containing 5% fetal bovine serum, with 50 μg/ml of gentamycin, 50 ng/ml of amphotericin B, 1 ng/ml of recombinant human EGF, and 1 ng/ml of recombinant human FGF.

Techniques: Cell Culture, Western Blot, Expressing, Control