Journal: Molecular Medicine Reports

Article Title: Iptakalim influences the proliferation and apoptosis of human pulmonary artery smooth muscle cells

doi: 10.3892/mmr.2016.5333

Figure Lengend Snippet: GLI reverses the effect on ET-1 induced change of apoptotic protein expression in HPASMCs by IPT (western blot, n=3). * P<0.05 vs. control group. # P<0.05 vs. ET-1 group. & P<0.05 vs. ET-1+IPT group. GLI, glibenclamide; HPASMC, human pulmonary artery smooth muscle cells; IPT, iptakalim.

Article Snippet: HPASMCs were purchased from Sciencell Research Laboratories (Carlsbad, CA, USA).

Techniques: Expressing, Western Blot, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Adenosine Monophosphate–Activated Protein Kinase Is Required for Pulmonary Artery Smooth Muscle Cell Survival and the Development of Hypoxic Pulmonary Hypertension

doi: 10.1165/rcmb.2012-0446OC

Figure Lengend Snippet: The activation of adenosine monophosphate–activated protein kinase (AMPK) is essential for the survival of human pulmonary artery smooth muscle cells (HPASMCs) during hypoxia. (A and B) HPASMCs were exposed to normoxia (N) (21% O2) or hypoxia (H) (3% O2) for 15 and 30 minutes (A) and 24 hours (B). The amounts of phospho-AMPK (pAMPK) at Thr-172 (T172) and total AMPK were determined, and the pAMPK/AMPK ratios were calculated. (C-F) HPASMCs were pretreated with dimethylsulfoxide (DMSO) or 10 μM Compound C (CC) for 1 hour, and then incubated under normoxia (N) or hypoxia (H) for 24 hours for the cell viability assay (C), 8 hours for the lactate dehydrogenase (LDH) assay (D), and for up to 5 hours to detect the cleavage of caspase-3 (E) and for a terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay (F). Data are expressed as means ± SEMs (n ≥ 3). *P < 0.05. **P < 0.01. Tubulin was used as loading control. CTL, control.

Article Snippet: HPASMCs were obtained from Lonza (Walkersville, MD) ( 22 ).

Techniques: Activation Assay, Incubation, Viability Assay, Lactate Dehydrogenase Assay, End Labeling, TUNEL Assay, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Adenosine Monophosphate–Activated Protein Kinase Is Required for Pulmonary Artery Smooth Muscle Cell Survival and the Development of Hypoxic Pulmonary Hypertension

doi: 10.1165/rcmb.2012-0446OC

Figure Lengend Snippet: The inhibition of AMPK α2 induces HPASMC apoptosis during hypoxia. (A) HPASMCs were transfected with small interfering RNA (siRNA) against AMPK α1 or α2 before exposure to normoxia (N) or hypoxia (H) for 6 hours, and LDH activity was determined as already described. Scrambled siRNA (si-Neg) was used as control. The amounts of AMPK α1 and α2 in the cell lysates were determined by Western blot analysis. β-actin was used as loading control. (B) Wild-type (WT), AMPK α1–null, and AMPK α2–null mouse embryo fibroblasts (MEFs) were exposed to normoxia or hypoxia for 8 hours, and then LDH activity was measured. (C) HPASMCs transfected with siRNA against AMPK α1 or α2 were incubated during normoxia or hypoxia for 8 hours, and then caspase-3 activity was measured. (D) MEFs were exposed to normoxia or hypoxia for 8 hours, and cell lysates were collected to measure caspase-3 activity. Data are expressed as means ± SEMs (n ≥ 3). *P < 0.05.

Article Snippet: HPASMCs were obtained from Lonza (Walkersville, MD) ( 22 ).

Techniques: Inhibition, Transfection, Small Interfering RNA, Activity Assay, Control, Western Blot, Incubation

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Adenosine Monophosphate–Activated Protein Kinase Is Required for Pulmonary Artery Smooth Muscle Cell Survival and the Development of Hypoxic Pulmonary Hypertension

doi: 10.1165/rcmb.2012-0446OC

Figure Lengend Snippet: Inhibition of AMPK α2 decreases the expression of prosurvival protein MCL-1, leading to HPASMC cell apoptosis during hypoxia. (A) HPASMCs were pretreated with dimethylsulfoxide (DMSO) or 10 μM Compound C (CC) for 1 hour, and then incubated under normoxia (N) or hypoxia (H) for 8 hours. The amount of prosurvival myeloid cell leukemia sequence 1 (MCL-1) and B-cell lymphoma-extra large (BCL-XL) and proapoptotic Bcl-2–associated death promoter (BAD) and BH3 interacting-domain death agonist (BID) in the cell lysates were determined by Western blot analysis. (B) Cells were transfected with siRNA against AMPK α1 or α2 (si-α1 or si-α2) and then exposed to normoxia (N) or hypoxia (H) for 6 hours. The amount of MCL-1 in the cell lysates was determined as described previously. Tubulin was used as loading control. The MCL-1/tubulin ratios are shown at the top. (C–E) HPASMCs were transfected with si–MCL-1 and exposed to normoxia (N) or hypoxia (H) for 6 hours. (C) The amount of MCL-1 in the cell lysates was determined by Western blot analysis, and the MCL-1/tubulin ratios are shown at the top. LDH activity (D) and caspase-3 activity (E) were determined as described previously. (F and G) HPASMCs were pretreated with various doses of obatoclax mesylate (GX 15-070) (F) and TW-37 (G) for 1 hour, and then incubated under normoxia (NMX) or hypoxia (HPX) for 24 hours. The HPASMC viability was measured as described previously. Data are expressed as means ± SEMs (n ≥ 3). *P < 0.05. **P < 0.01.

Article Snippet: HPASMCs were obtained from Lonza (Walkersville, MD) ( 22 ).

Techniques: Inhibition, Expressing, Incubation, Sequencing, Western Blot, Transfection, Control, Activity Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Adenosine Monophosphate–Activated Protein Kinase Is Required for Pulmonary Artery Smooth Muscle Cell Survival and the Development of Hypoxic Pulmonary Hypertension

doi: 10.1165/rcmb.2012-0446OC

Figure Lengend Snippet: AMPK α1 facilitates HPASMC survival during hypoxia by promoting autophagy. HPASMCs were either transfected with siRNA for AMPK α1 or α2 (A) or pretreated with dimethylsulfoxide (DMSO) or 1 mM 3-methyladenine (3-MA) for 1 hour (B), and then exposed to normoxia or hypoxia for 6 hours. The cleavage of microtubule-associated protein 1 light chain 3B (LC3B) was measured by Western blot analysis. β-actin (A) and tubulin (B) were used as loading controls. The LC3B-II/I ratios are shown at the top (B). After pretreatment with 3-MA and exposure to normoxia or hypoxia, LDH activity (C) and caspase-3 activity (D) were determined. Data are expressed as means ± SEMs (n ≥ 3). *P < 0.05. **P < 0.01.

Article Snippet: HPASMCs were obtained from Lonza (Walkersville, MD) ( 22 ).

Techniques: Transfection, Western Blot, Activity Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Adenosine Monophosphate–Activated Protein Kinase Is Required for Pulmonary Artery Smooth Muscle Cell Survival and the Development of Hypoxic Pulmonary Hypertension

doi: 10.1165/rcmb.2012-0446OC

Figure Lengend Snippet: Hypertensive human and mouse PASMCs express elevated levels of AMPK phosphorylation. (A) We compared the concentrations of phosphorylated and total AMPK in normal HPASMCs and in HPASMCs isolated from patients with pulmonary arterial hypertension (PAH). Representative blots are shown at the bottom, and the amounts of pAMPK and total AMPK are shown as the pAMPK/AMPK and AMPK/tubulin ratios at the top and middle, respectively. (B) Mice were exposed to normoxia (ambient air, N) or hypoxia (10% O2, H) for 3 weeks. Whole-lung homogenates were used to determine the concentrations of phosphorylated and total AMPK by Western blot analysis. Data are expressed as means ± SEMs (n ≥ 3). *P < 0.05. Tubulin was used as loading control. (C) The lung sections of mice described in B were immunolabeled with pAMPK and α–smooth muscle actin (SMA) antibodies and 4′6-diamidino-2-phenylindole (DAPI) for study, using fluorescence microscopy.

Article Snippet: HPASMCs were obtained from Lonza (Walkersville, MD) ( 22 ).

Techniques: Phospho-proteomics, Isolation, Western Blot, Control, Immunolabeling, Fluorescence, Microscopy

Journal: Frontiers in Cardiovascular Medicine

Article Title: Identification and validation of CCL5 as a key gene in HIV infection and pulmonary arterial hypertension

doi: 10.3389/fcvm.2024.1417701

Figure Lengend Snippet: Expression of CCL5 in hPAECs and hPASMCs. ( A ) Expression level of CCL5 between control and hypoxia groups in hPAECs. ( B ) Expression level of CCL5 between control and hypoxia groups in hPASMCs. hPAECs, human pulmonary artery endothelial cells; hPASMCs, human pulmonary artery smooth muscle cells. n = 3, * P < 0.05, ** P < 0.01.

Article Snippet: hPAECs were purchased from ScienCell (Shanghai, China, Cat. No. 3100). hPASMCs were obtained from Procell (Wuhan, China, Cat. No. CP-H243).

Techniques: Expressing, Control

Journal: Journal of Cardiovascular Pharmacology

Article Title: circNFXL1 Modulates the Kv2.1 Channel Function in Hypoxic Human Pulmonary Artery Smooth Muscle Cells via Sponging miR-29b-2-5p as a Competitive Endogenous RNA

doi: 10.1097/FJC.0000000000001396

Figure Lengend Snippet: circNFXL1 reversed the hypoxia-induced alterations in membrane potential and Kv currents in human pulmonary arterial smooth muscle cells (hPASMCs). A, circNFXL1 partially reversed the hypoxia-induced depolarization of resting membrane potential ( E m) in hPASMCs. The representative traces (B) and current-voltage relationships (IV) (C) of whole-cell Kv currents were recorded from hPASMCs in normoxia, hypoxia, and hypoxia+circNFXL1 conditions. D, The average amplitude of Kv currents recorded at +100 mV was compared between the 3 groups. * P < 0.05, ** P < 0.01, *** P < 0.001 (n = 10–12 cells). Mean ± SEM. Comparisons of data were acquired by one-way ANOVA followed by Bonferroni post-hoc. ANOVA, analysis of variance.

Article Snippet: Detection of intracellular Ca 2+ was conducted by imaging the fluorescence intensity of fluo-loaded hPASMCs (OLYMPUS, BX51).

Techniques:

Journal: Journal of Cardiovascular Pharmacology

Article Title: circNFXL1 Modulates the Kv2.1 Channel Function in Hypoxic Human Pulmonary Artery Smooth Muscle Cells via Sponging miR-29b-2-5p as a Competitive Endogenous RNA

doi: 10.1097/FJC.0000000000001396

Figure Lengend Snippet: circNFXL1-regulated Kv2.1 expression via miR-29b-2 under hypoxic conditions. A, qPCR and (B) WB analysis of Kv2.1 in hPASMCs transfected with circNFXL1 expression vector or circNFXL1+miR-29b-2 mix and cultured under hypoxic conditions for 24–48 hours (hypoxia/circNFXL1, hypoxia/circNFXL1 + miR29b). hPASMCs transfected with pLC5-ciR and cultured under normal/hypoxic conditions were used as controls (normoxia/hypoxia). ** P < 0.01(n = 5 individual experiments). Mean ± SEM. Comparisons of data were acquired by one-way ANOVA followed by Bonferroni post-hoc. ANOVA, analysis of variance.

Article Snippet: Detection of intracellular Ca 2+ was conducted by imaging the fluorescence intensity of fluo-loaded hPASMCs (OLYMPUS, BX51).

Techniques: Expressing, Transfection, Plasmid Preparation, Cell Culture

Journal: Journal of Cardiovascular Pharmacology

Article Title: circNFXL1 Modulates the Kv2.1 Channel Function in Hypoxic Human Pulmonary Artery Smooth Muscle Cells via Sponging miR-29b-2-5p as a Competitive Endogenous RNA

doi: 10.1097/FJC.0000000000001396

Figure Lengend Snippet: circNFXL1-regulated intracellular calcium via sponging miR-29b-2 under hypoxic conditions. Intracellular calcium level was measured using the Fluo-3-AM staining. The hPASMCs transfected with circNFXL1 expression vector or circNFXL1+miR-29b-2 mix and cultured under hypoxic conditions for 24–48 hours (hypoxia/circNFXL1, hypoxia/circNFXL1+miR29b). hPASMCs transfected with pLC5-ciR and cultured under normal/hypoxic conditions were used as controls (normoxia/hypoxia). After transfections, the cultured hPASMCs were labeled by Fura-3-AM. Detection of intracellular Ca 2+ was carried out by imaging the fluorescence intensity of fluo-loaded hPASMCs. * P < 0.05, ** P < 0.01 (n = 8–10 cells). Mean ± SEM. Comparisons of data were acquired by one-way ANOVA followed by Bonferroni post-hoc. ANOVA, analysis of variance.

Article Snippet: Detection of intracellular Ca 2+ was conducted by imaging the fluorescence intensity of fluo-loaded hPASMCs (OLYMPUS, BX51).

Techniques: Staining, Transfection, Expressing, Plasmid Preparation, Cell Culture, Labeling, Imaging, Fluorescence

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