Journal: Nature Communications

Article Title: MURC deficiency in smooth muscle attenuates pulmonary hypertension

doi: 10.1038/ncomms12417

Figure Lengend Snippet: ( a ) Representative immunostaining images of MURC, Cav1 and Cav3 in hPASMCs. Scale bar, 50 μm. ( b , c ) COS cells were transfected with pcDNA3.1-hMURC-HA and/or pcDNA3.1-T7-hCav1 ( b ) or pcDNA3.1-T7-hCav3 ( c ), and the cell lysates were immunoprecipitated with anti-HA and anti-T7 antibodies. ( d ) The in situ association of proteins in hPASMCs was assessed by the BiFC assay, which detects fluorescent signals in living cells when proteins associate with each other. Upper, hPASMCs were transfected with phmKGC-MC-hMURC and phmKGN-MC-hCav1. Lower, hPASMCs were transfected with phmKGN-MN-hMURC and phmKGC-MN-hCav3. Scale bar, 50 μm. Uncropped images of blots are shown in .

Article Snippet: GST-Cav1(FL), GST-Cav1(1–61), GST-Cav1(61–101), and GST-Cav1(135–178) were gifts from William C. Sessa (Addgene plasmid #22445, 22446, 22447, and 22448). hPASMCs were purchased from PromoCell GmbH (Germany).

Techniques: Immunostaining, Transfection, Immunoprecipitation, In Situ, Bimolecular Fluorescence Complementation Assay

Journal: Nature Communications

Article Title: MURC deficiency in smooth muscle attenuates pulmonary hypertension

doi: 10.1038/ncomms12417

Figure Lengend Snippet: ( a ) The proliferation capacity in hPASMCs transfected with control siRNA or MURC siRNA was assessed using a WST-1 cell proliferation assay system ( n =10 per group). * P <0.05 compared with control siRNA. ( b ) Migration in hPASMCs transfected with control siRNA or MURC siRNAs was assessed by a wound healing assay ( n =8 per group). Wound closure was quantified by the per cent change in the wound area. Scale bar, 200 μm. * P <0.05 compared with control siRNA. ( c , d ) Proliferation ( n =8 per group) and migration ( n =7–8 per group) capacities were assessed in MURC-overexpressing hPASMCs. Scale bar, 200 μm. * P <0.05 compared with LacZ. ( e ) RhoA activity was measured in hPASMCs transfected with control siRNA or MURC siRNAs ( n =6 per group). Starved cells were stimulated with 1% FBS for 1 h. * P <0.05 compared with control siRNA. ( f ) RhoA activity was measured in LacZ- and MURC-overexpressing hPASMCs ( n =3 per group). * P <0.05 compared with LacZ. Proliferation ( g ) and migration ( h ) capacities were assessed in hPASMCs incubated with or without a ROCK inhibitor, Y-27632 ( n =3–5 per group). Scale bar, 200 μm. ( i ) RhoA activity was assessed in hPASMCs transduced with LacZ and MURC with control siRNA or p115RhoGEF siRNAs ( n =8 per group). hPASMCs were infected with a puromycin-resistant retrovirus expressing LacZ and MURC-FLAG. After selection using puromycin, hPASMCs were transfected with control siRNA, p115RhoGEF siRNA-1, or p115RhoGEF siRNA-2. * P <0.05 compared with LacZ+control siRNA, † P <0.05 compared with MURC+control siRNA. Data are presented as mean±s.e.m.

Article Snippet: GST-Cav1(FL), GST-Cav1(1–61), GST-Cav1(61–101), and GST-Cav1(135–178) were gifts from William C. Sessa (Addgene plasmid #22445, 22446, 22447, and 22448). hPASMCs were purchased from PromoCell GmbH (Germany).

Techniques: Transfection, Proliferation Assay, Migration, Wound Healing Assay, Activity Assay, Incubation, Transduction, Infection, Expressing, Selection

Journal: Nature Communications

Article Title: MURC deficiency in smooth muscle attenuates pulmonary hypertension

doi: 10.1038/ncomms12417

Figure Lengend Snippet: ( a ) RhoA activity was assessed in hPASMCs transduced with LacZ, Cav1-HA and Cav1-HA and MURC-FLAG ( n =4 per group). hPASMCs were infected with a hygromycin-resistant retrovirus expressing LacZ and Cav1-HA. After selection using hygromycin, hPASMCs were infected with a puromycin-resistant retrovirus expressing LacZ and MURC-FLAG, and subsequently selected using puromycin. * P <0.05 compared with LacZ+LacZ, † P <0.05 compared with LacZ+Cav1. ( b ) COS cells were transfected with pcDNA3.1-hMURC-HA and/or pcDNA3.1-hGα13. The COS cell lysates incubated with GDP or GTPγS and GST pulldown was performed with GST-fusion Cav1 conjugated to glutathione-Sepharose beads. Precipitated proteins were blotted with anti-Gα13 and anti-GST antibodies ( n =3 per group). * P <0.05 compared with Gα13-GDP+Cav1, † P <0.05 compared with Gα13-GTPγS+Cav1. ( c ) The association of Gα13 with Cav1 was assessed in hPASMCs transfected with control siRNA or MURC siRNA ( n =3 per group). * P <0.05 compared with control siRNA. ( d ) GST-fusion Gα13 conjugated to glutathione-Sepharose beads was preloaded with GDP or GTPγS. GST pulldown was performed with COS cell lysates transfected with plasmids expressing the indicated proteins. Precipitated proteins were blotted with anti-FLAG and anti-GST antibodies ( n =5 per group). * P <0.05 compared with Gα13-GDP+p115RhoGEF, † P <0.05 compared with Gα13-GTPγS+p115RhoGEF, # P <0.05 compared with Gα13-GTPγS+p115RhoGEF+Cav1. ( e ) Each domain of Cav1 (FL: full length, 1–61: C-terminal domain, 61–101: oligomerization domain, 135–178: N-terminal domain) tagged with GST was conjugated to glutathione-Sepharose beads, and incubated with lysates from COS cells transfected with pcDNA3.1-hMURC-FLAG or pcDNA3.1-hGα13. Pulled-down protein was blotted with anti-MURC or anti-Gα13 antibodies. Arrows indicate each GST-fusion protein. Data are presented as mean±s.e.m. Uncropped images of blots are shown in .

Article Snippet: GST-Cav1(FL), GST-Cav1(1–61), GST-Cav1(61–101), and GST-Cav1(135–178) were gifts from William C. Sessa (Addgene plasmid #22445, 22446, 22447, and 22448). hPASMCs were purchased from PromoCell GmbH (Germany).

Techniques: Activity Assay, Transduction, Infection, Expressing, Selection, Transfection, Incubation

Journal: Nature Communications

Article Title: MURC deficiency in smooth muscle attenuates pulmonary hypertension

doi: 10.1038/ncomms12417

Figure Lengend Snippet: ( a ) Immunostaining images of MURC and p115RhoGEF in hPASMCs. Scale bar, 20 μm. ( b ) pcDNA3.1-hMURC-HA and/or pCS2FLAG-hp115RhoGEF were cotransfected into COS cells, and the cell lysates were then immunoprecipitated with anti-HA and anti-FLAG antibodies. ( c ) hPASMCs were transfected with phmKGN-MC-hMURC and phmKGC-MN-hp115RhoGEF. Scale bar, 50 μm. ( d ) COS cells were transfected with pcDNA3.1-hMURC-FLAG and/or pcDNA3.1-hRhoA-HA and cell lysates were immunoprecipitated with anti-FLAG and anti-HA antibodies. ( e ) Proposed roles of the MURC-mediated activation of Rho/ROCK signalling in PASMCs. When MURC is present, it associates with Cav1, leading to reductions in the association between Cav1 and Gα13, which facilitates the interaction of Gα13 with p115RhoGEF, thereby activating the Rho/ROCK pathway. MURC also serves as a platform to compartmentalize p115RhoGEF and RhoA at caveolae. In the absence of MURC, Cav1 interacts with Gα13 and inhibits the association of Gα13 with p115RhoGEF, leading to the suppression of the Rho/ROCK pathway. Uncropped images of blots are shown in .

Article Snippet: GST-Cav1(FL), GST-Cav1(1–61), GST-Cav1(61–101), and GST-Cav1(135–178) were gifts from William C. Sessa (Addgene plasmid #22445, 22446, 22447, and 22448). hPASMCs were purchased from PromoCell GmbH (Germany).

Techniques: Immunostaining, Immunoprecipitation, Transfection, Activation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: MiR‐629 regulates hypoxic pulmonary vascular remodelling by targeting FOXO3 and PERP

doi: 10.1111/jcmm.14385

Figure Lengend Snippet: The effects of hypoxia on miR‐629 expression, cell viability and cell proliferation in HPASMCs. A, The expression of miR‐629 in HPASMCs after being treated with hypoxia for 12, 24 and 48 h was determined by qRT‐PCR. B, Cell viability of HPASMCs after being treated with hypoxia for 12, 24 and 48 h was determined by CCK‐8 assay. C, Cell proliferation of HPASMCs after being treated with hypoxia for 12, 24 and 48 h was determined by BrdU assay. Data are expressed as mean ± standard deviation; n = 3. * P < 0.05, ** P < 0.01 and *** P < 0.001 versus control groups

Article Snippet: The HPASMCs were obtained from Sigma‐Aldrich (St. Louis, USA).

Techniques: Expressing, Quantitative RT-PCR, CCK-8 Assay, BrdU Staining, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: MiR‐629 regulates hypoxic pulmonary vascular remodelling by targeting FOXO3 and PERP

doi: 10.1111/jcmm.14385

Figure Lengend Snippet: Overexpression of miR‐629 promoted cell proliferation, cell migration and inhibited cell apoptosis of HPASMCs. A, The expression of miR‐629 in HPASMCs after being transfected with mimics NC or miR‐629 mimics was determined by qRT‐PCR. B, Cell viability and (C) cell proliferation of HPASMCs after being transfected with mimics NC or miR‐629 mimics was detected by CCK‐8 assay and BrdU assay respectively. (D and E) The expression of PCNA mRNA and protein in HPASMCs after being transfected with mimics NC or miR‐629 mimics were determined by qRT‐PCR and western blot respectively. (F) Cell migration of HPASMCs after being transfected with mimics NC or miR‐629 mimics was determined by transwell migration assay. (G and H) The caspase‐3 activity and the expression of caspase‐3 protein in HPASMCs after being transfected with mimics NC or miR‐629 mimics were determined by Caspase‐3 activity assay and western blot respectively. Data are expressed as mean ± standard deviation; n = 3. * P < 0.05, ** P < 0.01 and *** P < 0.001 versus control groups

Article Snippet: The HPASMCs were obtained from Sigma‐Aldrich (St. Louis, USA).

Techniques: Over Expression, Migration, Expressing, Transfection, Quantitative RT-PCR, CCK-8 Assay, BrdU Staining, Western Blot, Transwell Migration Assay, Activity Assay, Caspase-3 Activity Assay, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: MiR‐629 regulates hypoxic pulmonary vascular remodelling by targeting FOXO3 and PERP

doi: 10.1111/jcmm.14385

Figure Lengend Snippet: Knockdown of miR‐629 suppressed cell proliferation, cell migration and induced cell apoptosis of HPASMCs during hypoxia. A, The expression of miR‐629 in HPASMCs after being transfected with inhibitors NC or miR‐629 inhibitors was determined by qRT‐PCR. B, Cell viability and (C) cell proliferation of hypoxia‐treated HPASMCs after being transfected with inhibitors NC or miR‐629 inhibitors was detected by CCK‐8 assay and BrdU assay respectively. (D and E) The expression of PCNA mRNA and protein in hypoxia‐treated HPASMCs after being transfected with inhibitors NC or miR‐629 inhibitors were determined by qRT‐PCR and western blot respectively. F, Cell migration of hypoxia‐treated HPASMCs after being transfected with inhibitors NC or miR‐629 inhibitors was determined by transwell migration assay. (G and H) The caspase‐3 activity and the expression of caspase‐3 protein in hypoxia‐treated HPASMCs after being transfected with inhibitors NC or miR‐629 inhibitors were determined by Caspase‐3 activity assay and western blot respectively. Data are expressed as mean ± standard deviation; n = 3. * P < 0.05, ** P < 0.01 and *** P < 0.001 versus control groups

Article Snippet: The HPASMCs were obtained from Sigma‐Aldrich (St. Louis, USA).

Techniques: Migration, Expressing, Transfection, Quantitative RT-PCR, CCK-8 Assay, BrdU Staining, Western Blot, Transwell Migration Assay, Activity Assay, Caspase-3 Activity Assay, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: MiR‐629 regulates hypoxic pulmonary vascular remodelling by targeting FOXO3 and PERP

doi: 10.1111/jcmm.14385

Figure Lengend Snippet: miR‐629 directly targets FOXO3. A, The predicted binding sites between 3′UTR of FOXO3 and miR‐629 as determined by the targetscan software. B, The relative luciferase activity of the reporter vector containing wide‐type (WT) 3′UTR of FOXO3 was determined at 48 h after transfection with mimics NC or miR‐629 mimics. C, The relative luciferase activity of the reporter vector containing mutant (MUT) 3′UTR of FOXO3 was determined at 48 h after transfection with mimics NC or miR‐629 mimics. (D and E) The relative luciferase activity of the reporter vector containing mutant 3′UTR of FOXO3 was determined at 48 h after transfection with inhibitors NC or miR‐629 inhibitors in hypoxia‐treated HPASMCs. (F and G) The expression of FOXO3 mRNA and protein in HPASMCs after being transfected with mimics NC or miR‐629 mimics was determined by qRT‐PCR and western blot respectively. (H and I) The expression of FOXO3 mRNA and protein in HPASMCs after exposing to hypoxia or normoxia was determined by qRT‐PCR and western blot respectively. Data are expressed as mean ± standard deviation; n = 3. * P < 0.05, ** P < 0.01 and *** P < 0.001 versus control groups

Article Snippet: The HPASMCs were obtained from Sigma‐Aldrich (St. Louis, USA).

Techniques: Binding Assay, Software, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Mutagenesis, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: MiR‐629 regulates hypoxic pulmonary vascular remodelling by targeting FOXO3 and PERP

doi: 10.1111/jcmm.14385

Figure Lengend Snippet: MiR‐629 directly targets PERP. A, The predicted binding sites between 3′UTR of PERP and miR‐629 as determined by the targetscan software. B, The relative luciferase activity of the reporter vector containing wide‐type (WT) 3′UTR of PERP was determined at 48 h after transfection with mimics NC or miR‐629 mimics. C, The relative luciferase activity of the reporter vector containing mutant (MUT) 3′UTR of PERP was determined at 48 h after transfection with mimics NC or miR‐629 mimics. (D and E) The relative luciferase activity of the reporter vector containing mutant 3′UTR of PERP was determined at 48 h after transfection with inhibitors NC or miR‐629 inhibitors in hypoxia‐treated HPASMCs. (F and G) The expression of PERP mRNA and protein in HPASMCs after being transfected with mimics NC or miR‐629 mimics was determined by qRT‐PCR and western blot respectively. (H and I) The expression of PERP mRNA and protein in HPASMCs after exposing to hypoxia or normoxia was determined by qRT‐PCR and western blot respectively. Data are expressed as mean ± standard deviation; n = 3. * P < 0.05, ** P < 0.01 and *** P < 0.001 versus control groups

Article Snippet: The HPASMCs were obtained from Sigma‐Aldrich (St. Louis, USA).

Techniques: Binding Assay, Software, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Mutagenesis, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: MiR‐629 regulates hypoxic pulmonary vascular remodelling by targeting FOXO3 and PERP

doi: 10.1111/jcmm.14385

Figure Lengend Snippet: Overexpression of FOXO3 and PERP reversed the effects of miR‐629 overexpression on cell proliferation, migration and apoptosis of HPASMCs. (A and B) The expression of FOXO3 mRNA and protein in HPASMCs after being transfected with pcDNA3.1 or pcDNA3.1‐FOXO3 was determined by qRT‐PCR and western blot respectively. (C and D) The expression of PERP mRNA and protein in HPASMCs after being transfected with pcDNA3.1 or pcDNA3.1‐PERP was determined by qRT‐PCR and western blot respectively. (E) Cell viability and (F) cell proliferation of HPASMCs after being co‐transfected with miRNAs and plasmids were determined by CCK‐8 assay and BrdU assay respectively. G, The expression of PCNA mRNA in HPASMCs after being co‐transfected with miRNAs and plasmids was determined by qRT‐PCR. H, Cell migration of HPASMCs after being co‐transfected with miRNAs and plasmids was measured by transwell migration assay. I, Cell migration of HPASMCs after being co‐transfected with miRNAs and plasmids was measured by transwell migration assay. Data are expressed as mean ± standard deviation; n = 3. * P < 0.05, ** P < 0.01 and *** P < 0.001 versus control groups

Article Snippet: The HPASMCs were obtained from Sigma‐Aldrich (St. Louis, USA).

Techniques: Over Expression, Migration, Expressing, Transfection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, BrdU Staining, Transwell Migration Assay, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: MiR‐629 regulates hypoxic pulmonary vascular remodelling by targeting FOXO3 and PERP

doi: 10.1111/jcmm.14385

Figure Lengend Snippet: Overexpression of FOXO3 and PERP reversed the hypoxia‐induced effects on cell proliferation, migration and apoptosis of HPASMCs. HPASMCs under the normoxia condition served as negative controls. In other groups, HPASMCs were treated with hypoxia for 48 h, (A) cell viability, (B) cell proliferation, (C) expression of PCNA mRNA, (D) cell migration and (E) caspase‐3 activity were determined by respective in vitro functional assays at 24 h post‐transfection with pcDNA3.1, pcDNA3.1‐FOXO3 or pcDNA3.1‐PERP. Data are expressed as mean ± standard deviation; n = 3. * P < 0.05, ** P < 0.01 and *** P < 0.001 versus control groups

Article Snippet: The HPASMCs were obtained from Sigma‐Aldrich (St. Louis, USA).

Techniques: Over Expression, Migration, Expressing, Activity Assay, In Vitro, Functional Assay, Transfection, Standard Deviation

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 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: 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