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: HPASMCs (4–8 passages, ScienCell Research Laboratories, USA) were cultured in complete smooth muscle cell medium (SMCM) .

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: HPASMCs (4–8 passages, ScienCell Research Laboratories, USA) were cultured in complete smooth muscle cell medium (SMCM) .

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: HPASMCs (4–8 passages, ScienCell Research Laboratories, USA) were cultured in complete smooth muscle cell medium (SMCM) .

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

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: Expression Profiles of circRNAs and Identification of hsa_circ_0007608 and hsa_circ_0064656 as Potential Biomarkers for COPD-PH Patients

doi: 10.2147/COPD.S424712

Figure Lengend Snippet: qRT-PCR validation of circRNAs expression in serum and pulmonary vascular cell. ( A and B ) Two significantly up-regulated circRNAs in serum, n=12. ( C ) hsa_circ_0007608 was constantly up-regulated with the prolongation of hypoxia in HPAECs, n=3. ( D and E ) hsa_circ_0064656 decreased significantly in HPAECs, while increased in HPASMCs, n=3. (*P<0.05, **P<0.01, *** P<0.001).

Article Snippet: Pulmonary arterial smooth muscle cells (HPASMCs) were purchased from iCell Research Laboratory cultured in smooth muscle culture medium.

Techniques: Quantitative RT-PCR, Biomarker Discovery, Expressing

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: Expression Profiles of circRNAs and Identification of hsa_circ_0007608 and hsa_circ_0064656 as Potential Biomarkers for COPD-PH Patients

doi: 10.2147/COPD.S424712

Figure Lengend Snippet: Identification and validation of hub mRNAs. ( A ) Correlation heat map of 35 key mRNA. ( B ) The protein–protein interaction (PPI) of key mRNAs.( C – E ) VCAM1, VCAN and THBS1 were up-regulated in HPAECs after 48 hours of hypoxia treatment, n=3 (**P<0.01, *** P<0.001). ( F – H ) VCAM1, VCAN and THBS1 were up-regulated in HPASMCs after 48 hours of hypoxia treatment, n=3 (*** P<0.001). ( I and J ) Representative immunohistochemistry staining and quantitative analysis of VCAN in two groups of human lung tissue, ×200, scale bar: 100μm, n=3. (***P< 0.001, normal vs COPD-PH group, Student t test).

Article Snippet: Pulmonary arterial smooth muscle cells (HPASMCs) were purchased from iCell Research Laboratory cultured in smooth muscle culture medium.

Techniques: Biomarker Discovery, Immunohistochemistry, Staining

Journal: Experimental & Molecular Medicine

Article Title: USP15 promotes pulmonary vascular remodeling in pulmonary hypertension in a YAP1/TAZ-dependent manner

doi: 10.1038/s12276-022-00920-y

Figure Lengend Snippet: a Identification of hPASMCs by immunofluorescence staining. Scale bar: 100 μm. b , c The mRNA and protein levels of USP15 in the hypoxia-treated hPASMCs. d The protein levels of t-YAP1 and t-TAZ in the hypoxia-treated hPASMCs. e The protein levels of n-YAP1 and n-TAZ in the hypoxia-treated hPASMCs. Data are represented as the mean ± SD, n = 4 in each group. Compared with values at 0 h, * P < 0.05, ** P < 0.01.

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were purchased from iCell Bioscience, Inc., Shanghai. hPASMCs were cultured in specific medium (PriMed-iCell-004-LS, iCell Bioscience, China) at 37 °C in a humidified incubator with 5% CO 2 . hPASMCs at passages 2-7 were used for further study.

Techniques: Immunofluorescence, Staining

Journal: Experimental & Molecular Medicine

Article Title: USP15 promotes pulmonary vascular remodeling in pulmonary hypertension in a YAP1/TAZ-dependent manner

doi: 10.1038/s12276-022-00920-y

Figure Lengend Snippet: Adenovirus delivering shRNA against hUSP15 was used to downregulate USP15 expression in hPASMCs. After 48 h of infection, cells were cultured under hypoxia for another 24 h. The protein level ( a ) and mRNA level ( b ) of USP15 in the treated cells were measured by western blotting and real-time PCR, respectively. c The abundance of PCNA protein was detected by western blot analysis. d A CCK-8 assay was performed to determine cell viability after treatments. e , f The protein levels of t-YAP1, t-TAZ, n-YAP1, and n-TAZ in the treated cells were measured by western blotting. g , h Cell migration in each group was determined by wound healing assay. Scale bar: 200 μm. Data are represented as the mean ± SD, n = 4 in each group.

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were purchased from iCell Bioscience, Inc., Shanghai. hPASMCs were cultured in specific medium (PriMed-iCell-004-LS, iCell Bioscience, China) at 37 °C in a humidified incubator with 5% CO 2 . hPASMCs at passages 2-7 were used for further study.

Techniques: shRNA, Expressing, Infection, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, CCK-8 Assay, Migration, Wound Healing Assay

Journal: Experimental & Molecular Medicine

Article Title: USP15 promotes pulmonary vascular remodeling in pulmonary hypertension in a YAP1/TAZ-dependent manner

doi: 10.1038/s12276-022-00920-y

Figure Lengend Snippet: Adenovirus delivering overexpressed hUSP15 was used to upregulate USP15 expression in hPASMCs under normoxia. Western blot analysis ( a ) and real-time PCR ( b ) were conducted to determine the infectivity of Ad-hUSP15-OV after 72 h of infection. Afterward, hPASMCs were coinfected with adenovirus delivering overexpressed hUSP15 and hYAP1-sh or hTAZ-sh. c The expression level of PCNA in hPASMCs was detected by immunoblot analysis after coinfection. d A CCK-8 assay was performed to detect the viability of hPASMCs after coinfection. e , f The expression levels of t-YAP1, t-TAZ, n-YAP1, and n-TAZ in hPASMCs were detected by western blotting after coinfection. g , h Cell migration in hPASMCs was determined by wound healing assays after coinfection. Scale bar: 200 μm. Data are represented as the mean ± SD, n = 4 in each group. Compared to the Vector+NCsh-2 group, # P < 0.05, ## P < 0.01; compared to the hUSP15-OV + NCsh-2 group, * P < 0.05, ** P < 0.01.

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were purchased from iCell Bioscience, Inc., Shanghai. hPASMCs were cultured in specific medium (PriMed-iCell-004-LS, iCell Bioscience, China) at 37 °C in a humidified incubator with 5% CO 2 . hPASMCs at passages 2-7 were used for further study.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Infection, CCK-8 Assay, Migration, Plasmid Preparation

Journal: Experimental & Molecular Medicine

Article Title: USP15 promotes pulmonary vascular remodeling in pulmonary hypertension in a YAP1/TAZ-dependent manner

doi: 10.1038/s12276-022-00920-y

Figure Lengend Snippet: a Anti-USP15 immunoprecipitation was used to determine the interaction between USP15 and YAP1 or TAZ in the control and hypoxic hPASMCs. b Anti-YAP1 immunoprecipitation to determine the interaction between YAP1 and USP15 in the control or hypoxic hPASMCs. c Anti-TAZ immunoprecipitation to determine the interaction between TAZ and USP15 in the control or hypoxic hPASMCs. d hPASMCs were infected with Ad-hUSP15-sh or Ad-NCsh-2 for 48 h. After 3 h of starvation, cells were exposed to hypoxia for 24 h and treated with CHX (25 μg/ml) or MG132 (5 μM) for 0, 1, 4, or 8 h (at the last 8 h of hypoxic time). Cell samples were harvested for immunoblot analysis to detect YAP1 expression. The percentage of YAP1/actin is shown in the right panel. e hPASMCs infected with Ad-hUSP15-OV or Ad-Vector were treated with MG132 (5 μM) under normoxia for 8 h, and cells were harvested for immunoprecipitation using anti-YAP1 antibody. f Under normoxia, infected hPASMCs were subjected to a CHX pulse-chase assay. Cell samples were harvested for immunoblot analysis to detect YAP1 expression. The percentage of YAP1/Actin is shown in the right panel. g His-tagged USP15 and Myc-tagged YAP1 were cotransfected into HEK293T cells. At 48 h post-transfection, cell samples were harvested for immunoprecipitation using an anti-His antibody. h HEK293T cells were cotransfected with Myc-tagged YAP1 and His-tagged USP15/vector. At 48 h post-transfection, cells were treated with CHX (25 μg/ml) for 0, 1, 4, and 8 h. YAP1 expression in cell samples was determined by western blot analysis using an anti-Myc antibody. The percentage of YAP1/actin is shown in the right panel. i His-tagged USP15, HA-tagged Ubi and Myc-tagged YAP1 were cotransfected into HEK293T cells. At 48 h post-transfection, cells were treated with MG132 (5 μM) for 5 h. Cell samples were then harvested for immunoprecipitation using anti-Myc antibody. Data are represented as the mean ± SD, n = 4 in each group.

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were purchased from iCell Bioscience, Inc., Shanghai. hPASMCs were cultured in specific medium (PriMed-iCell-004-LS, iCell Bioscience, China) at 37 °C in a humidified incubator with 5% CO 2 . hPASMCs at passages 2-7 were used for further study.

Techniques: Immunoprecipitation, Control, Infection, Western Blot, Expressing, Plasmid Preparation, Pulse Chase, Transfection

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Rapamycin inhibits mTORC1 and mTORC2. (A) hPASMCs were treated with 100 nM rapamycin for the indicated times and analyzed by immunoblotting for the proteins level of p-p70S6k, p70S6k, p-AKT (S473), p-AKT (T308), AKT. (B) immunoblotting analyses of p-p70S6k, p70S6k, p-AKT (S473), and AKT in hPASMCs, which were stimulated with 5 μg/ml insulin for 24h before treatment with 100 nM rapamycin. (C) hPASMCs were treated with 100 nM rapamycin for the indicated times, and then cell lysates were prepared for and immunoprecipitation (IP) with mTOR antibody. The elution from IP was analyzed by immunoblotting for the levels of mTOR and Rictor. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. NS means not significant. *** p < 0.001; ** p < 0.01; * p < 0.05 versus control.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Western Blot, Immunoprecipitation, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Imatinib inhibits phosphorylation of PDGFRα/β induced by rapamycin in hPASMCs. (A) hPASMCs were treated with 100 nM rapamycin for the indicated times and analyzed by immunoblotting for the proteins level of p-PDGFRα/β, PDGFRα, PDGFRβ. (B) Immunoblotting analyses of p-PDGFRα/β, PDGFRα, PDGFRβ, p-AKT (S473), p-AKT (T308), p-S6 and S6 in hPASMCs treated with vehicle (Control), 100 nM rapamycin (Rap), 5 uM imatinib (Ima) and 100 nM rapamycin + 5 uM imatinib (Rap + Ima) for 48 h. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. NS means not significant. *** p < 0.001, ** p < 0.01, * p < 0.05 versus control.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Effects of rapamycin combined with imatinib on the viability, proliferation and migration of hPASMCs. (A) Cell viability was determined by measuring the absorbance at 0, 24, 48 and 72 h after different drug treatments. (B) A scratch was applied to cell monolayers, and migration of the cells towards the wound was recorded by photomicrographs at 0, 4, and 8h ( n = 3); summarized data showing percent wound closure [(0h wound area–4h or 8h wound area)/0h wound area] * 100%. (C) BrdU assay was performed to determine hPASMCs proliferation under normoxia and hypoxia (3% 0 2 ) for 24 and 48 h. (D) BrdU assay was performed to determine hPASMCs proliferation at 24 and 48 h after different drug treatments. Data are presented as the mean ± SE. Two-way ANOVA was used for statistical analysis. *** p < 0.001; ** p < 0.01; * p < 0.05 versus control; ### p < 0.001, ## p < 0.01, # p < 0.05 versus Rap; $$$ p < 0.001, $$ p < 0.01, $ p < 0.05 versus Ima.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Migration, BrdU Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Rapamycin combined with imatinib attenuates PASMC proliferation and remodeling induced by MCT. (A) H&E staining in lung tissue sections. Summarized data showing pulmonary artery media wall thickness. (B) Lung sections were stained α-SMA (red) and PCNA (green). Yellow arrowheads point at PCNA positive PASMCs and white arrowheads show the vessels. For each of the 5 groups, approximately 600 PASMC nuclei and 100 fields were analyzed. Summarized data showing PCNA positive cells and muscularization. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. *** p < 0.001, ** p < 0.01, * p < 0.05 versus control; ### p < 0.001, ## p < 0.01, # p < 0.05 versus MCT with vehicle; $$$ p < 0.001, $$ p < 0.01, $ p < 0.05 versus MCT with rapamycin.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Rapamycin combined with imatinib attenuates PASMC proliferation and remodeling induced by Hypoxia/Sugen. (A) H&E staining of lung tissue sections and summarized data showing pulmonary artery media wall thickness. (B) Lung sections were stained with α-SMA (red) and PCNA (green). Yellow arrowheads point at PCNA positive PASMCs and white arrowheads show the vessels. For each of the 5 groups, approximately 600 PASMC nuclei and 100 fields were analyzed. Summarized data showing PCNA positive cells and muscularization. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. NS means no significant. *** p < 0.001, ** p < 0.01, * p < 0.05 versus control; ### p < 0.001, ## p < 0.01, # p < 0.05 versus Hypoxia/Sugen with vehicle; $$$ p < 0.001, $$ p < 0.01, $ p < 0.05 versus Hypoxia/Sugen with rapamycin.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Effects of rapamycin combined with imatinib on mTOR and PDGFR signaling pathways in pulmonary artery. (A) Pulmonary artery vessels of were isolated for protein extraction, and the expression of mTORC 1, mTORC 2 and PDGFR signaling pathway related proteins were detected by immunoblotting. Data are presented as the mean ± SE. One-way ANOVA followed by Graphpad prism was used for statistical analysis. NS means no significant. *** p < 0.001; ** p < 0.01; * p < 0.05 versus control. ### p < 0.001; ## p < 0.01; # p < 0.05 versus MCT with vehicle. (B) The schematic representation of the findings of this study: rapamycin chronic treatment in hPASMCs induced the highly expression of phosphorylation of PDGFRs. Imatinib inhibits phosphorylation of PDGFRα/β induced by rapamycin. Abbreviations: GF, growth factors; RTK, receptor tyrosine kinase; PDGF, platelet derived growth factor; PDGFR, platelet derived growth factor receptor; PI3K, phosphoatidylinositol 3-kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-bisphosphate; mTORC1, mTOR complex 1; mTORC2, mTOR complex 2.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Protein-Protein interactions, Isolation, Protein Extraction, Expressing, Western Blot, Control, Phospho-proteomics, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Sildenafil protects against pulmonary hypertension induced by hypoxia in neonatal rats via activation of PPARγ-mediated downregulation of TRPC

doi: 10.3892/ijmm.2021.5074

Figure Lengend Snippet: Sildenafil attenuated the hypoxia-induced downregulation of PPARγ expression and inhibited the hypoxia-induced upregulation of TRPC and Ki67 expression in HPASMCs. (A) HPASMCs were identified by immunofluorescence staining for α-SMA in cells grown under normoxic conditions. (B) Western blot and (C) RT-qPCR analysis of the increase in PPARγ protein and mRNA expression in HPASMCs induced by different concentrations of sildenafil under hypoxic conditions. Each of the four groups were treated with 0, 1, 10 or 50 nM sildenafil. (D) Western blot and (E) RT-qPCR analysis of the sildenafil-mediated attenuation of hypoxia-induced downregulation of PPARγ expression and sildenafil-mediated inhibition of hypoxia-induced upregulation of TRPC and Ki67 expression in HPASMCs. There were three experimental groups: i) The normoxic control group (60 h), ii) the hypoxia group (60 h, 4% O 2 ), and iii) the hypoxia + sildenafil group (60 h, 4% O 2 , 50 nM sildenafil). Data are presented as the mean ± standard deviation of three repeats. * P<0.05 vs. control. PPARγ, peroxisome proliferator-activated receptor γ; HPASMC, human pulmonary artery smooth muscle cell; SMA, smooth muscle actin; TRPC, transient receptor potential canonical; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: HPASMCs derived from ScienCell primary cells were used for the in vitro experiments.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Inhibition, Control, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Sildenafil protects against pulmonary hypertension induced by hypoxia in neonatal rats via activation of PPARγ-mediated downregulation of TRPC

doi: 10.3892/ijmm.2021.5074

Figure Lengend Snippet: A PPARγ inhibitor (GW9662) inhibited the sildenafil-induced downregulation of TRPC and Ki67 protein expression in HPASMCs under hypoxic conditions. (A) Western blot and (B) RT-qPCR analysis of the inhibitory effects of different concentrations of a PPARγ inhibitor (GW9662) on PPARγ protein expression in HPASMCs under hypoxic conditions. Cells were treated with either 0, 1 or 10 nM GW9662. (C) Western blot and (D) RT-qPCR analysis of the inhibitory effect of a PPARγ inhibitor (GW9662) on the sildenafil-induced downregulation of TRPC and Ki67 protein expression in HPASMCs under hypoxic conditions. There were four groups: i) The hypoxic control group (60 h, 4% O 2 ), ii) the hypoxia + sildenafil group (60 h, 4% O 2 ), iii) the hypoxia + GW9662 group (60 h, 4% O 2 , 10 nM), and (iv) the hypoxia + sildenafil + GW9662 group (60 h, 4% O 2 , 10 nM). Data are presented as the mean ± standard deviation of three repeats. * P<0.05 vs. control, # P>0.05 vs. control. PPARγ, peroxisome proliferator-activated receptor γ; HPASMC, human pulmonary artery smooth muscle cell; TRPC, transient receptor potential canonical; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: HPASMCs derived from ScienCell primary cells were used for the in vitro experiments.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Sildenafil protects against pulmonary hypertension induced by hypoxia in neonatal rats via activation of PPARγ-mediated downregulation of TRPC

doi: 10.3892/ijmm.2021.5074

Figure Lengend Snippet: si-PPARγ reversed the sildenafil-induced downregulation of TRPC and Ki67 expression under hypoxic conditions. (A) Western blot and (B) RT-qPCR analysis of the downregulation of PPARγ protein expression in HPASMCs transfected with si-PPARγ under normoxic conditions. There were four groups: si-NC, siR-PP1, siR-PP2 and siR-PP3. (C) Western blot and (D) RT-qPCR analysis of the reversal of the sildenafil-induced downregulation of TRPC and Ki67 protein expression in HPASMCs induced by PPARγ siRNA under hypoxic conditions. There were four groups: i) The hypoxic control group (60 h, 4% O 2 ), ii) the hypoxia + sildenafil group (60 h, 4% O 2 ), iii) the hypoxia + siR-PP1 group (60 h, 4% O 2 ), and iv) the hypoxia + sildenafil + siR-PP1 group. Data are presented as the mean ± standard deviation of three repeats. * P<0.05 vs. control. PPARγ, peroxisome proliferator-activated receptor γ; HPASMC, human pulmonary artery smooth muscle cell; siRNA, small interfering RNA; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: HPASMCs derived from ScienCell primary cells were used for the in vitro experiments.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Control, Standard Deviation, Small Interfering RNA, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The European Respiratory Journal

Article Title: Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension

doi: 10.1183/13993003.01698-2021

Figure Lengend Snippet: Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1) activation in experimental and human pulmonary arterial hypertension (PAH). a) Representative immunofluorescence micrograph of human lung sections from control and idiopathic PAH (IPAH) patients. Staining was undertaken for Pin1 (green) and vessel identity was visualised using α-smooth muscle actin (SMA) (red). Scale bar=50 µm. b, d) Protein expression of Pin1 in smooth muscle cells (control n=4, IPAH n=9) and endothelial cells (control n=4, IPAH n=5) isolated from pulmonary arteries of control and IPAH patients. Regulation at protein level was analysed using Western blot analysis followed by c, e) densitometric analysis. f–i) Correlation of Pin1 with clinical characteristics of IPAH patients, such as mean pulmonary arterial pressure (mPAP) (n=4, r=0.8177, p=0.0468), pulmonary capillary wedge pressure (n=6, r= −8825, p=0.1175), cardiac index (n=4, r= −0.8276, p=01724) and systolic pulmonary artery pressure (n=7, r= −0.6285, p=0.1306), respectively. j, l) Western blot analysis of Pin1 in lung homogenates exposed to Sugen5416/hypoxia (SuHx) (normoxia (NOX) n=4, SuHx n=4) and chronic hypoxia (HOX), respectively (NOX n=6, HOX n=7) followed by k, m) densitometric analysis. Pan-actin is taken as loading control. DAPI: 4′,6-diamidino-2-phenylindole; hPASMCs: human pulmonary artery smooth muscle cells; hPAECs: human pulmonary artery endothelial cells; ns : nonsignificant; A.U.: arbitrary unit. *: p<0.05, ***: p<0.001 (t-test).

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were either obtained from the Universities of Giessen and Marburg Lung Center Giessen Biobank, member of DZL Platform Biobanking or purchased from Lonza (Basel, Switzerland).

Techniques: Activation Assay, Immunofluorescence, Control, Staining, Expressing, Isolation, Western Blot

Journal: The European Respiratory Journal

Article Title: Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension

doi: 10.1183/13993003.01698-2021

Figure Lengend Snippet: Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1) blockage results in the suppression of vascular cell proliferation in vitro . a) Human pulmonary artery smooth muscle cells (hPASMCs) from controls and idiopathic pulmonary arterial hypertension (IPAH) patients cultured in SmGM-2 were serum-starved and treated with Juglone or dimethyl sulfoxide (DMSO) (vehicle) in the presence of platelet-derived growth factor (PDGF)-BB for 24 h. b) Representative Western blots of Pin1 and proliferating cell nuclear antigen (PCNA) expression in control and IPAH hPASMCs followed by c) densitometric analysis 24 h after Pin1 mRNA knockdown. Immunofluorescence staining for Ki-67 + cells in d) Pin1-silenced (si) and f) Juglone-exposed hPASMCs. g) Human pulmonary artery endothelial cells (hPAECs) were serum-starved (0.2% fetal bovine serum (FBS) in M200) and stimulated with 10% FBS with or without Juglone for 24 h. Proliferation of Pin1-silenced e) hPASMCs and h) hPAECs of donor control and IPAH patients in presence or absence of e) PDGF-BB and h) 10% FBS determined by 5-bromo-2-deoxyuridine (BrdU) incorporation. The rate of DNA synthesis for a, e, g and h) was examined by measuring of BrdU incorporation [ A 370 nm]. Scr: scrambled; ns : nonsignificant. Statistical analysis was performed using one-way ANOVA with Newman–Keuls post hoc test for multiple comparisons. **: p<0.01, ***: p<0.001, ****: p<0.0001 versus PDGF-BB or 10% FBS treated cells; # : p<0.05, ## : p<0.01, ### : p<0.001, #### : p<0.0001 versus si scrambled or dimethyl sulfoxide (DMSO)-treated cells; § : p<0.05, §§ : p<0.01, §§§ : p<0.001, §§§§ : p<0.0001 versus si scrambled treated or IPAH cells. Data from three independent experiments are presented as mean± sem .

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were either obtained from the Universities of Giessen and Marburg Lung Center Giessen Biobank, member of DZL Platform Biobanking or purchased from Lonza (Basel, Switzerland).

Techniques: In Vitro, Cell Culture, Derivative Assay, Western Blot, Expressing, Control, Knockdown, Immunofluorescence, Staining, BrdU Incorporation Assay, DNA Synthesis

Journal: The European Respiratory Journal

Article Title: Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension

doi: 10.1183/13993003.01698-2021

Figure Lengend Snippet: Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1) blockage results in initiation of cell apoptosis in vitro . Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay after 24 h treatment with increasing concentration of Juglone of a) control and i) idiopathic pulmonary arterial hypertension (IPAH) human pulmonary artery smooth muscle cells (hPASMCs), and of e) control and o) IPAH human pulmonary artery endothelial cells (hPAECs). b, j, m) Representative Western blots and c, d, k, l, n) subsequent densitometric analysis of control and IPAH hPASMCs after Juglone treatment. f, p, s) Representative Western blots and g, h, q, r, t) subsequent densitometric analysis of control and IPAH hPAECs. PARP: poly (ADP-ribose) polymerase; PCNA: proliferating cell nuclear antigen. *: p<0.05; **: p<0.01; ***: p<0.001 versus dimethyl sulfoxide (DMSO)-treated control cells. Statistical analysis was performed using one-way ANOVA with Newman–Keuls post hoc test for multiple comparisons. Data from three independent experiments are presented as mean± sem .

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were either obtained from the Universities of Giessen and Marburg Lung Center Giessen Biobank, member of DZL Platform Biobanking or purchased from Lonza (Basel, Switzerland).

Techniques: In Vitro, TUNEL Assay, Concentration Assay, Control, Western Blot

Journal: The European Respiratory Journal

Article Title: Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension

doi: 10.1183/13993003.01698-2021

Figure Lengend Snippet: Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1) controls the activity of multitude of transcription factors. a) Control and idiopathic pulmonary arterial hypertension (IPAH) human pulmonary artery smooth muscle cells (hPASMCs) after 24 h of serum starvation were subjected to platelet-derived growth factor (PDGF)-BB (50 ng·mL −1 ), epidermal growth factor (EGF) (5 ng·mL −1 ) and growth medium (GM) with 5% fetal bovine serum (FBS). Intracellular Pin1 levels were monitored by ELISA. *: p<0.05, ****: p<0.0001 versus control PASMCs; ## : p<0.01, #### : p<0.0001 versus IPAH hPASMCs; §§ : p<0.01 IPAH hPASMCs versus control hPASMCs. Statistical analysis was performed using one-way ANOVA with Newman–Keuls post hoc test for multiple comparisons. Data from three independent experiments are presented as mean± sem . b) Pin1-silenced and Juglone-treated hPASMCs were stimulated with GM for 24 h and nuclear protein extracts were used for transcription factor activation profile array, presented as log-transformed signals in a volcano plot. c) Log-transformed scatter plot of combined transcription factor activation/inactivation in Pin1-silenced and Juglone-treated hPASMCs. Data from two independent experiments are presented. d, f) Western blots and e, g) subsequent densitometry analyses of hypoxia-inducible factor (HIF)-1α and C/EBPα transcription factors in Pin1-silenced control and IPAH hPASMCs subjected to hypoxia for 24 h. h) Hypoxia-responsive element (HRE) luciferase activity in Pin1-silenced hPASMCs after 24 h of hypoxia. Scr: scrambled; ns : nonsignificant. *: p<0.05; ****: p<0.0001 for normoxia (NOX) si Scr versus hypoxia (HOX) si Scr; § : p<0.05; §§§§ : p<0.0001 for HOX si Scr versus HOX si Pin1. Data from three independent experiments are presented as mean± sem .

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were either obtained from the Universities of Giessen and Marburg Lung Center Giessen Biobank, member of DZL Platform Biobanking or purchased from Lonza (Basel, Switzerland).

Techniques: Activity Assay, Control, Derivative Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Transformation Assay, Western Blot, Luciferase