Journal: Journal of Smooth Muscle Research

Article Title: Glucose-6-phosphate dehydrogenase and MEG3 controls hypoxia-induced expression of serum response factor (SRF) and SRF-dependent genes in pulmonary smooth muscle cell

doi: 10.1540/jsmr.58.34

Figure Lengend Snippet: G6PD inhibitor and MEG-3 siRNA regulates Srf expression in human pulmonary arterial SMCs. A) MEG3 expression level is decreased by MEG3-siRNA (50 nmol/l) in human pulmonary arterial SMCs exposed to normoxia (Nx; 21% O 2 ) and hypoxia (Hx; 3% O 2 ). Administration of 4091 (1 µmol/l) to human pulmonary arterial SMCs cultured under Nx or Hx upregulated MEG3 expression and this was blocked by MEG3-siRNA (50 nmol/l). There were no differences in expression of MEG3 between the application of control-siRNA with and without 4091. B) Srf expression in human SMCs exposed to Nx or Hx and treated with 4091 (1 µmol/l) and control (Ctrl)- or MEG3-siRNA (50 nmol/l). N=5–6. Statistical analysis was performed using the one-way ANOVA. Sidak’s test was used to compare multiple groups after ANOVA analysis.

Article Snippet: Human pulmonary artery smooth muscle cells (SMCs) were purchase from Lonza (MD, USA).

Techniques: Expressing, Cell Culture, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Regulation of Bcl-xLExpression in Lung Vascular Smooth Muscle

doi: 10.1165/rcmb.2006-0359oc

Figure Lengend Snippet: Figure 2. Effects of Bcl-xL on pulmonary vas- cular SMC apoptosis. (A) BPASMC were in- fected with adenovirus expressing Bcl-xL (AdBcl-xL) for 48 h, then treated with SNP (300 M) for 17 h. Cells were washed with PBS, trypsinized, incubated with Trypan Blue, and the number of viable cells was counted on a hematocytometer. The values represent means SE. Letters (a and b) denote that the values with the same letter are significantly different from each other at P 0.05. Top panel shows the expression level of Bcl-xL with or without adenovirus (adv)-mediated over- expression. (B) BPASMC were infected with AdBcl-xL or control adenovirus (AdCont) for 48 h, then treated with SNP (100 M) for 17 h. Mitochondrial membrane potentials were measured by DePsipher Kit. In healthy mitochondria, the DePsipher dye aggregates and forms red fluorescence. When the mem- brane potential is disrupted during the early stage of apoptosis, the dye cannot cross the mitochondrial membrane and is visualized as a monomeric form with green fluorescence in the cytosol. Fluorescence signals were ob- tained using a red/green dual filter, and un- der these conditions, green fluorescence was not observed in cells without staining with the dye. Green apoptotic cells are indicated by the arrows. (C) BPASMC were pretreated with 5-HT or ET-1 for 2 h, then treated with SNAP (100 M) or DNR (2 M) for 24 h. Percent of apoptotic cells were monitored by neutral comet assay. Values represent means SE. The values denoted by the same letter (a, b, or c) are significantly different from each other at P 0.05.

Article Snippet: Bovine pulmonary artery SMC (BPASMC) (19) from mid-size pulmonary arteries and human pulmonary artery SMC (HPASMC) (Cell Applications, San Diego, CA) at 2–6 passages were maintained in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.5% fungisone at 5% CO2 and 37 C. Cells were treated with SNP (Sigma Chemical, St. Louis, MO), S-nitroso-N-acetylpenicillamine (SNAP; Calbiochem, San Diego, CA), daunorubicin (DNR; Sigma), serotonin (5-hydroxytryptamine, 5-HT; Sigma) and endothelin-1 (ET-1; Sigma) in media supplemented only with antibiotics.

Techniques: Expressing, Incubation, Over Expression, Infection, Control, Membrane, Fluorescence, Staining, Neutral Comet Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Regulation of Bcl-xLExpression in Lung Vascular Smooth Muscle

doi: 10.1165/rcmb.2006-0359oc

Figure Lengend Snippet: Figure 3. Effects of SNP on Bcl-xL expression. (A) Rats were subjected to chronic hypoxia with 10% O2 in an OxyCycler Oxygen Profiler for 2 wk to elicit pulmonary vascular remodeling. Remodeled pulmonary arteries were surgically isolated, cut into 2-mm segments, and placed in Dulbecco’s modified Eagle’s medium (DMEM). Arterial segments were treated with SNP (300 M) for 20 h. Tissues were homogenized in Trizol and total RNA was prepared. RT-PCR was performed with primers for bcl-xL and g3pdh mRNA. The bar graph represents means SE of the intensity of the bcl-xL band expressed in arbitrary units (a.u.). (B) BPASMC were treated with SNP for 24 h at various concentra- tions. Cell lysates (10 g protein) were subjected to Western blot to monitor levels of Bcl-xL. ERK was used as a loading control. The bar graph represents means SE of the intensity of Bcl-xL band from cells untreated or treated with 100 M SNP (n 3). (C) BPASMC were transfected with the luciferase gene controlled by the 0.6-kb bcl-xL promoter region without (open circles) or with (solid triangles) SNP. Cell lysates were prepared at various time points after transfection and luciferase activities were measured and expressed in resonance units (R.U.). (D) Cells were co-transfected with firefly luciferase gene con- trolled by 0.6-kb bcl-xL promoter (pBcl-xL) and Renilla luciferase con- trolled by thymidine kinase promoter (pTK), and treated with various concentrations of SNP. Cell lysates were prepared and luciferase activi- ties were measured. The values represent means SE of the ratio of firefly luciferase activity to Renilla luciferase activity. Asterisk denotes a significantly different value compared with control at P 0.05.

Article Snippet: Bovine pulmonary artery SMC (BPASMC) (19) from mid-size pulmonary arteries and human pulmonary artery SMC (HPASMC) (Cell Applications, San Diego, CA) at 2–6 passages were maintained in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.5% fungisone at 5% CO2 and 37 C. Cells were treated with SNP (Sigma Chemical, St. Louis, MO), S-nitroso-N-acetylpenicillamine (SNAP; Calbiochem, San Diego, CA), daunorubicin (DNR; Sigma), serotonin (5-hydroxytryptamine, 5-HT; Sigma) and endothelin-1 (ET-1; Sigma) in media supplemented only with antibiotics.

Techniques: Expressing, Isolation, Modification, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Transfection, Luciferase, Activity Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Regulation of Bcl-xLExpression in Lung Vascular Smooth Muscle

doi: 10.1165/rcmb.2006-0359oc

Figure Lengend Snippet: Figure 4. Effects of SNP on GATA-4. (A) BPASMC were treated with SNP for 20 h. Nuclear extracts were prepared and the GATA DNA-binding activity was moni- tored by EMSA. The bar graph represents means SE of the intensity of GATA activ- ity from cells untreated or treated with SNP (300 M) (n 3). Asterisk denotes a value significantly different from the control value at P 0.05. (B) BPASMC were infected with control adenovirus (AdCont) or adenovirus expressing wild- type GATA-4 (AdGATA4) for 24 h, then treated with SNP (100 M) for 24 h. Cell lysates were prepared and levels of Bcl-xL expression were monitored by Western blot. The ERK antibody was used as a load- ing control. The values in the bar graph represent means SE. Asterisk denotes values significantly different from SNP- treated values at P 0.05. (C) HPASMC were treated with SNP (100 M) for dura- tions indicated. Total RNA was isolated and mRNA expression levels of gata4 and g3pdh were determined by RT-PCR. The bar graph shows the ratio of gata4 to g3pdh bands. Similar results were ob- tained in three separate experiments. (D) Rats were subjected to chronic hypoxia with 10% O2 in an OxyCycler Oxygen Profiler for 2 wk to elicit pulmonary vascu- lar remodeling. Remodeled pulmonary arteries were surgically isolated, cut into 2-mm segments, and plated in DMEM. Arterial segments were treated with SNP (300 M) for 20 h. Arterial segments were homogenized in Trizol and total RNA was prepared. RT-PCR was performed with primers for gata4 and g3pdh mRNA. The bar graph represents means SE of the intensity of the gata4 band. Asterisk de- notes a value significantly different from the control value at P 0.05.

Article Snippet: Bovine pulmonary artery SMC (BPASMC) (19) from mid-size pulmonary arteries and human pulmonary artery SMC (HPASMC) (Cell Applications, San Diego, CA) at 2–6 passages were maintained in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.5% fungisone at 5% CO2 and 37 C. Cells were treated with SNP (Sigma Chemical, St. Louis, MO), S-nitroso-N-acetylpenicillamine (SNAP; Calbiochem, San Diego, CA), daunorubicin (DNR; Sigma), serotonin (5-hydroxytryptamine, 5-HT; Sigma) and endothelin-1 (ET-1; Sigma) in media supplemented only with antibiotics.

Techniques: Binding Assay, Activity Assay, Control, Infection, Expressing, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Regulation of Bcl-xLExpression in Lung Vascular Smooth Muscle

doi: 10.1165/rcmb.2006-0359oc

Figure Lengend Snippet: Figure 5. Effects of SNP on the gata4 gene promoter activity. (A) A scheme depicting the mouse gata4 gene structure with the major tran- scriptional start site of mouse gata4 gene identified by 5RACE to occur 4.1 kb upstream of the translational start site. The 1,000 bp upstream from the identified transcriptional start site (shaded area) that is con- served among various species was cloned into a luciferase reporter vector. (B) BPASMC were co-transfected with the firefly luciferase con- struct controlled by the 1,000-bp proximal region of the gata4 promoter (pGATA4) and Renilla luciferase construct controlled by the thymidine kinase promoter (pTK). Cells were then treated with SNP (300 M) for 24 h, cell lysates were prepared, and luciferase activities were measured. Values represent means SE of the ratio of pGATA4-luciferase to pTK luciferase activities (n 4). Asterisk denotes values significantly different from the untreated control value at P 0.05. (C) The region of the gata4 promoter 1,000 bp proximal to the transcriptional start site was truncated to generate regions of the promoter 500 or 250 bp upstream from the transcriptional start site. BPASMC were transfected with lucifer- ase constructs controlled by these regions of the gata4 promoter. Values represent means SE of the ratio of pGATA4-luciferase and pTK lucifer- ase activities (n 6–11). (D) BPASMC were co-transfected with the firefly luciferase construct controlled by the 1,000, 500 or 250 bp fragment of the gata4 promoter and pTK-Renilla luciferase construct. Cells were then treated with SNP (200 M) for 24 h, cell lysates were prepared, and luciferase activities were measured. Values represent means SE of percent of the ratio of pGATA4-luciferase and pTK luciferase activities relative to untreated controls.

Article Snippet: Bovine pulmonary artery SMC (BPASMC) (19) from mid-size pulmonary arteries and human pulmonary artery SMC (HPASMC) (Cell Applications, San Diego, CA) at 2–6 passages were maintained in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.5% fungisone at 5% CO2 and 37 C. Cells were treated with SNP (Sigma Chemical, St. Louis, MO), S-nitroso-N-acetylpenicillamine (SNAP; Calbiochem, San Diego, CA), daunorubicin (DNR; Sigma), serotonin (5-hydroxytryptamine, 5-HT; Sigma) and endothelin-1 (ET-1; Sigma) in media supplemented only with antibiotics.

Techniques: Activity Assay, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Construct, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Regulation of Bcl-xLExpression in Lung Vascular Smooth Muscle

doi: 10.1165/rcmb.2006-0359oc

Figure Lengend Snippet: Figure 6. Effects of SNP on transcription factors which bind to the proximal 250 bp gata4 promoter. (A) The sequence of the 250-bp gata4 promoter proximal to the transcriptional start site. Putative binding sites for transcription factors are in- dicated. (B) Nuclear extracts were prepared from untreated BPASMC, and the DNA- binding activity toward the 32P-labeled dou- ble-stranded 250 bp gata4 promoter probe was monitored by EMSA. Supershift experi- ments were performed with antibodies (ab) indicated. No ab indicates controls for supershift experiments without the in- clusion of any antibodies in nuclear ex- tracts from untreated cells. The letter A indicates the DNA-protein complex with- out supershift. The letter B indicates the free probe. The gel at the bottom shows that the band A can be eliminated by in- creasing amounts of the cold 250-bp gata4 promoter probe. (C) BPASMC were treated with SNP (100 or 300 M) for 2 h, and nuclear extracts were prepared. The DNA-binding activity toward the

Article Snippet: Bovine pulmonary artery SMC (BPASMC) (19) from mid-size pulmonary arteries and human pulmonary artery SMC (HPASMC) (Cell Applications, San Diego, CA) at 2–6 passages were maintained in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.5% fungisone at 5% CO2 and 37 C. Cells were treated with SNP (Sigma Chemical, St. Louis, MO), S-nitroso-N-acetylpenicillamine (SNAP; Calbiochem, San Diego, CA), daunorubicin (DNR; Sigma), serotonin (5-hydroxytryptamine, 5-HT; Sigma) and endothelin-1 (ET-1; Sigma) in media supplemented only with antibiotics.

Techniques: Sequencing, Binding Assay, Activity Assay, Labeling