Journal: Scientific Reports

Article Title: Follistatin-like 1 protects against hypoxia-induced pulmonary hypertension in mice

doi: 10.1038/srep45820

Figure Lengend Snippet: ( a ) Serum concentration of FSTL1 protein by ELISA in patients with COPD only (n = 8), COPD combined with PH (n = 8) and healthy controls (CTL, n = 7). ( b ) Effect of chronic hypoxia on RVSP and RVHI ( c ) in C57BL/6 mice. n = 8. ( d ) QRT-PCR analysis of Fstl1 mRNA in lung tissue of C57BL/6 mice under hypoxia as normalized by Gapdh mRNA. n = 10. ( e ) Representative cropped western blots and statistical analysis of FSTL1 protein in lung tissue of C57BL/6 mice under hypoxia as normalized by GAPDH. n = 10. ( f ) Serum concentration of FSTL1 protein by ELISA in C57BL/6 mice under hypoxia. n = 7–11. ( g ) Representative immunofluorescence images showing FSTL1 (green) and α-SMA (red) staining of pulmonary arterioles from lung sections in hypoxia-treated mice and untreated ones. Nuclei were stained with DAPI (blue). n = 4–5. Bar = 50 μm. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01. COPD = chronic obstructive pulmonary diseases. PH = pulmonary hypertension. RVSP = right ventricular systolic pressure. RVHI = right ventricular hypertrophy index. W = week. ELISA = enzyme-linked immunosorbent assay. GAPDH = glyceraldehyde-3-phosphate dehydrogenase. α-SMA = α-smooth muscle actin. DAPI = 4′,6-diamidino-2-phenylindole.

Article Snippet: For systemic delivery of FSTL1 protein, mice were randomly subjected to tail-vein injection with recombinant human FSTL1 protein (100 ng/g mouse , R & D Systems, USA) or with equal dosage of vehicle (PBS), immediately before the beginning of hypoxia or after 2 weeks of hypoxia, and repeated every 3 days until the end.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Follistatin-like 1 protects against hypoxia-induced pulmonary hypertension in mice

doi: 10.1038/srep45820

Figure Lengend Snippet: ( a ) Representative cropped western blots of FSTL1 protein in Fstl1 +/− and WT mice as normalized by GAPDH. n = 6. ( b ) RVSP in Fstl1 +/− and WT mice under hypoxia. n = 7–12. ( c ) RVHI in Fstl1 +/− and WT mice under hypoxia. n = 8–10. ( d ) Representative images showing hematoxylin and eosin staining of pulmonary arterioles from lung sections in Fstl1 +/− and WT mice under hypoxia. n = 4–5. Bar = 20 μm. ( e ) Representative immunofluorescence images showing α-SMA staining (red) of pulmonary arterioles from lung sections in Fstl1 +/− and WT mice under hypoxia. n = 4–5. Bar = 50 μm. ( f ) MT% of pulmonary arteries grouped by 0–50 μm and 50–100 μm in outer diameter from lung sections in Fstl1 +/− and WT mice under hypoxia. n = 5. ( g ) Numbers of completely muscularized arterioles (0–50 μm in outer diameter) per 10 fields from lung sections in Fstl1 +/− and WT mice under hypoxia. n = 5. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. HPH = hypoxia-induced PH. WT = wide type. RVSP = right ventricular systolic pressure. RVHI = right ventricular hypertrophy index. MT = media thickness. W = week. GAPDH = glyceraldehyde-3-phosphate dehydrogenase. α-SMA = α-smooth muscle actin.

Article Snippet: For systemic delivery of FSTL1 protein, mice were randomly subjected to tail-vein injection with recombinant human FSTL1 protein (100 ng/g mouse , R & D Systems, USA) or with equal dosage of vehicle (PBS), immediately before the beginning of hypoxia or after 2 weeks of hypoxia, and repeated every 3 days until the end.

Techniques: Western Blot, Staining, Immunofluorescence

Journal: Scientific Reports

Article Title: Follistatin-like 1 protects against hypoxia-induced pulmonary hypertension in mice

doi: 10.1038/srep45820

Figure Lengend Snippet: ( a ) FSTL1 treatment regimen in HPH model of mice. ( b ) Representative cropped western blots of serum FSTL1 protein in mice intravenously administrated with FSTL1 or PBS under hypoxia. n = 4. RVSP ( c ) and RVHI ( d ) in mice intravenously administrated with FSTL1 or PBS under hypoxia. n = 5. ( e ) Representative images showing hematoxylin and eosin staining of pulmonary arterioles from lung sections in mice intravenously administrated with FSTL1 or PBS under hypoxia. n = 4–5. Bar = 20 μm. (f) Representative immunofluorescence images showing α-SMA staining (red) of pulmonary arterioles from lung sections in mice intravenously administrated with FSTL1 or PBS under hypoxia. n = 4–5. Bar = 50 μm. (g) MT% of pulmonary arteries grouped by 0–50 μm and 50–100 μm in outer diameter from lung sections in mice intravenously administrated with FSTL1 or PBS under hypoxia. n = 5. ( h ) Numbers of completely muscularized arterioles (0–50 μm in outer diameter) per 10 fields from lung sections in mice intravenously administrated with FSTL1 or PBS under hypoxia. n = 5. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. HPH = hypoxia-induced PH. PBS = phosphate buffer saline. RVSP = right ventricular systolic pressure. RVHI = right ventricular hypertrophy index. α-SMA = α-smooth muscle actin. MT = media thickness. W = week.

Article Snippet: For systemic delivery of FSTL1 protein, mice were randomly subjected to tail-vein injection with recombinant human FSTL1 protein (100 ng/g mouse , R & D Systems, USA) or with equal dosage of vehicle (PBS), immediately before the beginning of hypoxia or after 2 weeks of hypoxia, and repeated every 3 days until the end.

Techniques: Western Blot, Staining, Immunofluorescence, Saline

Journal: Scientific Reports

Article Title: Follistatin-like 1 protects against hypoxia-induced pulmonary hypertension in mice

doi: 10.1038/srep45820

Figure Lengend Snippet: ( a ) Effect of FSTL1 on cellular viability under hypoxia and PDGF-BB ( b ) in MTT assay. n = 3. ( c ) Effect of FSTL1 on DNA synthesis under hypoxia and PDGF-BB ( d ) in BrdU assay for flow cytometer analysis. Cells in synthesis phrase (S, P4) at a cell cycle was calculated as the percent of P4/(P3 + P4 + P5). n = 3. ( e ) Effect of FSTL1 on cellular migration in transwell chamber. Nuclei of trans-membrane cells were stained with DAPI (blue). n = 3. Bar = 50 μm. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. HPASMCs = human pulmonary artery smooth muscle cells. P3 = G0/G1 phrase. P4 = S phase. P5 = G2 phrase. P6 = apoptosis phrase. MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. BrdU = 5-bromo-2-deoxyuridine. DAPI = 4’,6-diamidino-2-phenylindole. N = normoxia. H = hypoxia. V = vehicle. P = PDGF-BB. PDGF = platelet derived growth factor. FSTL1 = ng/mL.

Article Snippet: For systemic delivery of FSTL1 protein, mice were randomly subjected to tail-vein injection with recombinant human FSTL1 protein (100 ng/g mouse , R & D Systems, USA) or with equal dosage of vehicle (PBS), immediately before the beginning of hypoxia or after 2 weeks of hypoxia, and repeated every 3 days until the end.

Techniques: MTT Assay, DNA Synthesis, BrdU Staining, Flow Cytometry, Migration, Membrane, Staining, Derivative Assay

Journal: Scientific Reports

Article Title: Follistatin-like 1 protects against hypoxia-induced pulmonary hypertension in mice

doi: 10.1038/srep45820

Figure Lengend Snippet: ( a ) QRT-PCR analysis of FSTL1 mRNA in HPASMCs transfected with siRNA or N.C., as normalized by GAPDH mRNA. n = 4. ( b ) Representative cropped western blots of FSTL1 protein in HPASMCs transfected with siRNA or N.C. n = 3. ( c ) Effect of FSTL1 siRNA transfection on cellular viability in MTT assay. n = 3. ( d ) Effect of FSTL1 siRNA transfection on DNA synthesis in BrdU assay for flow cytometer analysis. Cells in synthesis phrase (S, P4) at a cell cycle was calculated as the percent of P4/(P3 + P4 + P5). n = 3. ( e ) Effect of FSTL1 siRNA transfection on cellular migration in transwell chamber. Nuclei of trans-membrane cells were stained with DAPI (blue). n = 3. Bar = 50 μm. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. SiRNA = small interfering RNA. N.C. = negative control. P3 = G0/G1 phrase. P4 = S phase. P5 = G2 phrase. P6 = apoptosis phrase. N = normoxia. H = hypoxia. GAPDH = glyceraldehyde-3-phosphate dehydrogenase. DAPI = 4′, 6-diamidino-2-phenylindole. MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. BrdU = 5-bromo-2-deoxyuridine.

Article Snippet: For systemic delivery of FSTL1 protein, mice were randomly subjected to tail-vein injection with recombinant human FSTL1 protein (100 ng/g mouse , R & D Systems, USA) or with equal dosage of vehicle (PBS), immediately before the beginning of hypoxia or after 2 weeks of hypoxia, and repeated every 3 days until the end.

Techniques: Quantitative RT-PCR, Transfection, Western Blot, MTT Assay, DNA Synthesis, BrdU Staining, Flow Cytometry, Migration, Membrane, Staining, Small Interfering RNA, Negative Control

Journal: Scientific Reports

Article Title: Follistatin-like 1 protects against hypoxia-induced pulmonary hypertension in mice

doi: 10.1038/srep45820

Figure Lengend Snippet: ( a ) Representative cropped western blots and statistical analysis of phosphorylations of AMPK (p-AMPK, n = 5) and ERK (p-ERK, n = 6) in lung tissue of Fstl1 +/− mice and WT controls under hypoxia. ( b ) Representative cropped western blots and statistical analysis of p-AMPK (n = 5) and p-ERK (n = 5) in lung tissue of mice treated with FSTL1 or PBS under hypoxia. Representative cropped western blots and statistical analysis of p-ERK ( c ) and p-AMPK ( d ) in HPASMCs exposed to hypoxia or normoxia for 24 h. n = 3. ( e ) Representative cropped western blots and statistical analysis of p-ERK in HPASMCs transfected with FSTL1 siRNA exposed to hypoxia or normoxia for 24 h. n = 3. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. HPH = hypoxia-induced PH. ERK = extracellular regulated kinase. AMPK = AMP-activated protein kinase. PBS = phosphate buffer saline. FSTL1 = 250 ng/mL. SiRNA = small interfering RNA. W = week.

Article Snippet: For systemic delivery of FSTL1 protein, mice were randomly subjected to tail-vein injection with recombinant human FSTL1 protein (100 ng/g mouse , R & D Systems, USA) or with equal dosage of vehicle (PBS), immediately before the beginning of hypoxia or after 2 weeks of hypoxia, and repeated every 3 days until the end.

Techniques: Western Blot, Transfection, Saline, Small Interfering RNA

Journal: Scientific Reports

Article Title: Follistatin-like 1 protects against hypoxia-induced pulmonary hypertension in mice

doi: 10.1038/srep45820

Figure Lengend Snippet: ( a ) Effect of ERK inactivation on FSTL1 knockdown-induced cellular viability under hypoxia in MTT assay. n = 3. ( b ) Effect of ERK inactivation on FSTL1 knockdown-induced DNA synthesis under hypoxia in BrdU assay for flow cytometer analysis. Cells in synthesis phrase (S, P4) at a cell cycle was calculated as the percent of P4/(P3 + P4 + P5). n = 3. ( c ) Effect of ERK inactivation on FSTL1 knockdown-induced cellular migration under hypoxia in transwell chamber. Nuclei of trans-membrane cells were stained with DAPI (blue). n = 3. Bar = 50 μm. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. ERK = extracellular regulated kinase. P3 = G0/G1 phrase. P4 = S phase. P5 = G2 phrase. P6 = apoptosis phrase. DAPI = 4′,6-diamidino-2-phenylindole. BrdU = 5-bromo-2-deoxyuridine. SiRNA = small interfering RNA. MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Article Snippet: For systemic delivery of FSTL1 protein, mice were randomly subjected to tail-vein injection with recombinant human FSTL1 protein (100 ng/g mouse , R & D Systems, USA) or with equal dosage of vehicle (PBS), immediately before the beginning of hypoxia or after 2 weeks of hypoxia, and repeated every 3 days until the end.

Techniques: Knockdown, MTT Assay, DNA Synthesis, BrdU Staining, Flow Cytometry, Migration, Membrane, Staining, Small Interfering RNA