Journal: Cardiovascular Research

Article Title: Carfilzomib reverses pulmonary arterial hypertension

doi: 10.1093/cvr/cvw047

Figure Lengend Snippet: CFZ-induced cell killing is ubiquitin-dependent. HPASMCs were transfected with control siRNA and Ub siRNA. (A) Knocking down efficiency was monitored by western blotting. (B) After transfection for 48 h, the transfection medium was changed to the starvation medium. Four hours later, cells were treated with CFZ (0.3 μM) for 20 h. The cell number was determined by counting on a haemocytometer. (C and D) Cell lysates were subjected to immunoblotting to analyse the expression of p62 and Bcl-xL. (E) Cell lysates were immunoprecipitated (IP) with TP53INP1 antibody, followed by immunoblotting with the LC3B antibody. The bar graphs represent means ± SEM. *Values significantly different from each other at P< 0.05 (n = 4).

Article Snippet: Human PASMCs (HPASMCs; ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in accordance with the manufacturer's instructions in 5% CO 2 at 37°C.

Techniques: Ubiquitin Proteomics, Transfection, Control, Western Blot, Expressing, Immunoprecipitation

Journal: Cardiovascular Research

Article Title: Carfilzomib reverses pulmonary arterial hypertension

doi: 10.1093/cvr/cvw047

Figure Lengend Snippet: CFZ induces autophagic cell death. (A and B) Pulmonary arteries (PAs) were surgically isolated from rats subjected to SU5416/hypoxia treatment with remodelled PA and from normal rats, homogenized, and subjected to western blotting analysis for LC3B-II and p62 expression. Bar graphs represent means ± SEM. *Values significantly different from each other at P< 0.05. (Control, n = 6; Control + CFZ, n = 6; PAH, n = 5; PAH + CFZ, n = 6). (C–E) HPASMCs were transfected with control siRNA and LC3B, AMPK or TP53INP1 siRNA. After 48 h, transfection medium were changed to starvation medium for 4 h, and then HPASMCs were treated with CFZ (0.3 μM) for 20 h. Bar graphs show the cell number determined by counting on a haemocytometer (LC3B siRNA, n = 6; AMPK siRNA, n = 4; TP53INP1, n = 4). *Values significantly different from each other at P< 0.05. (F) HPASMCs were treated with CFZ (0.3 μM) for 20 h, and cell lysates were immunoprecipitated (IP) with TP53INP1 antibody, followed by immunoblotting with the LC3B antibody. The bar graph represents means ± SEM. *Values significantly different from each other at P< 0.05(n = 3).

Article Snippet: Human PASMCs (HPASMCs; ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in accordance with the manufacturer's instructions in 5% CO 2 at 37°C.

Techniques: Isolation, Western Blot, Expressing, Control, Transfection, Immunoprecipitation

Journal: Cardiovascular Research

Article Title: Carfilzomib reverses pulmonary arterial hypertension

doi: 10.1093/cvr/cvw047

Figure Lengend Snippet: CFZ induces protein ubiquitination. (A) Isolated pulmonary arteries from the SU5416/hypoxia model of PAH were homogenized, and subjected to western blotting to monitor protein ubiquitination using a ubiquitin (Ub) antibody. (Control, n = 6; Control + CFZ, n = 6; PAH, n = 5; PAH + CFZ, n = 6). (B) Protein ubiquitination was monitored in isolated pulmonary arteries from the SU5416/ovalbumin model of PAH. (PAH, n = 6; PAH + CFZ, n = 6). Bar graphs represent means ± SEM. *Values significantly different from each other at P< 0.05. (C) HPASMCs were treated with various doses of CFZ for 20 h. Cell lysates were analysed for protein ubiquitination by western blotting. (D) Cells were treated with CFZ (0.3 μM) for various durations. Bar graphs represent means ± SEM. *Values significantly different from untreated control at P< 0.05 (n = 6).

Article Snippet: Human PASMCs (HPASMCs; ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in accordance with the manufacturer's instructions in 5% CO 2 at 37°C.

Techniques: Ubiquitin Proteomics, Isolation, Western Blot, Control

Journal: Cardiovascular Research

Article Title: Carfilzomib reverses pulmonary arterial hypertension

doi: 10.1093/cvr/cvw047

Figure Lengend Snippet: Identifications of proteins that are ubiquitinated by CFZ. (A) HPASMCs were treated with CFZ (0.3 μM) for 20 h, and cell lysates were immunoprecipitated (IP) with the Ub antibody. Bands indicated by arrows in Coomassie Blue-stained gels were consistently increased by CFZ treatment. The bar graph represents means ± SEM (n = 3). Mass spectrometry identified that these bands contain MVP and HSP90. Ubiquitination of MVP was confirmed in (B) CFZ-treated HPASMCs (n = 6), (C) isolated pulmonary arteries from SU5416/ovalbumin model (n = 4), and (D) isolated pulmonary arteries from SU5416/hypoxia model (n = 4) by immunoprecipitation with Ub antibody followed by immunoblotting with MVP. The ubiquitination of HSP90 were confirmed in (E) CFZ-treated HPASMCs (n = 6), (F) isolated pulmonary arteries from SU5416/ovalbumin model (n = 6), and (G) isolated pulmonary arteries from SU5416/hypoxia model (n = 4) by immunoprecipitation with Ub antibody followed by immunoblotting with HSP90 antibody. (H and I) HPASMCs were transfected with siRNA to knockdown MVP or HSP90. Efficiency of siRNA knockdown is shown in the western blotting data. Cells were treated with CFZ (0.3 μM) for 20 h, and the cell number was determined by counting on a haemocytometer. (MVP siRNA, n = 5; HSP90 siRNA, n = 6). *Values significantly different from each other at P< 0.05.

Article Snippet: Human PASMCs (HPASMCs; ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in accordance with the manufacturer's instructions in 5% CO 2 at 37°C.

Techniques: Immunoprecipitation, Staining, Mass Spectrometry, Ubiquitin Proteomics, Isolation, Western Blot, Transfection, Knockdown

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: Astragaloside IV blocks monocrotaline-induced pulmonary arterial hypertension by improving inflammation and pulmonary artery remodeling

doi: 10.3892/ijmm.2020.4813

Figure Lengend Snippet: ASIV attenuates hypoxia-induced HPAEC dysfunction. (A) TNF-α concentration in HPAEC culture media. (B) IL-1β concentration in HPAEC culture media. (C) Western blot analysis of HIF-1α and VEGF protein levels in HPAECs under normoxic or hypoxic conditions. (D) HIF-1α and VEGF protein levels, respectively (relative to normoxia group). Values are the means ± SEM of 3 independent experiments. * P<0.01 compared with the normoxia group; # P<0.05; ## P<0.01, compared with the hypoxia group. ASIV, astragaloside IV; HPAEC, human pulmonary artery endothelial cell; N, normoxia; H, hypoxia.

Article Snippet: Primary human PAECs (HPAECs) and human PASMCs (HPASMCs) were obtained from ScienCell Research Laboratories.

Techniques: Concentration Assay, Western Blot