Journal: Physiological Reports

Article Title: The effect of mitoTEMPO on the development of hypoxia‐induced pulmonary hypertension in male mice

doi: 10.14814/phy2.70804

Figure Lengend Snippet: Effect of mitoTEMPO treatment on HIF‐1α stabilization in vitro. HIF‐1α protein levels were assessed by western blot, and steady‐state mRNA levels for lactate dehydrogenase A ( Ldha ) and pyruvate dehydrogenase kinase 1 ( Pdk1 ) by quantitative real‐time PCR in (a) CMT167 cells, (b) hPASMCs, and (c) mPASMCs, exposed to normoxia (21% O 2 ), severe hypoxia (1% O 2 ), or mild hypoxia (10% O 2 ) for 24 h and treated with triphenylphosphonium (TPP + ) (blue dots) or mitoTEMPO (MT) (red dots). Immunoblots shown are representative of three independent experiments. CMT167: mouse lung carcinoma epithelial cells; hPASMCs: human pulmonary artery smooth muscle cells; mPASMCs: mouse pulmonary artery smooth muscle cells. Densitometric analysis of HIF‐1α bands normalized to β‐Actin. Data are presented as mean ± SD. Statistical comparisons were made using two‐way ANOVA with Tukey's post hoc test ( n = 3 per group). (ns: no signal). Quantitative real‐time PCR data reflect mean ΔCt ± SD ( n = 3 per experimental group).

Article Snippet: Mouse lung carcinoma epithelial (CMT167) cells (10032302, Merck, Germany) and human PASMCs (hPASMCs) (C‐12521, PromoCell, Germany) were purchased.

Techniques: In Vitro, Western Blot, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Opsin 3 and 4 mediate light-induced pulmonary vasorelaxation that is potentiated by G protein-coupled receptor kinase 2 inhibition

doi: 10.1152/ajplung.00091.2017

Figure Lengend Snippet: Expression of photorelaxation proteins in rat pulmonary arteries (PAs), rat pulmonary arterial smooth muscle cells (PASMCs), and human PASMCs. A: Opsin 3 (Opn3) and Opsin 4 (Opn4) were both detected in PAs, but Opsin 5 (Opn5) was not. B: G protein-coupled receptor kinase 2 (GRK2) was detected in rat PA (n = 5). Expression of Opn3, Opn4, and GRK2 in isolated rPASMCs via qRT-PCR (C) and Western blot (D) (n = 5) is shown. E: immunofluorescence images of hPASMCs stained for Opn3, Opn4, or GRK2 (n = 5). F and G: RT-PCR and qRT-PCR of hPASMC mRNA showing expression of Opn3, Opn4, and GRK2 but not Opn5 (n = 4–5). H: Western blot of hPASMC lysates tested for Opn3, Opn4, and GRK2 (n = 5) (+, with reverse transcriptase, RT; -, no RT). ***P < 0.001.

Article Snippet: Human PASMCs (hPASMCs; PromoCell, Heidelberg, Germany) were maintained in smooth muscle cell growth medium 2 (PromoCell) in a humidified incubator at 37°C and 5% CO 2 and were used for experiments between passages 3 and 7 .

Techniques: Expressing, Isolation, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Opsin 3 and 4 mediate light-induced pulmonary vasorelaxation that is potentiated by G protein-coupled receptor kinase 2 inhibition

doi: 10.1152/ajplung.00091.2017

Figure Lengend Snippet: G protein-coupled receptor kinase 2 (GRK2) desensitizes the photorelaxation response and interacts directly with Opn3 and Opn4. A: repeated blue light (455 nm) stimulation on rat pulmonary arteries (PAs) with or without GRK2 inhibitor (n = 5). Attenuation in photorelaxation was observed in vessels not treated with GRK2 inhibitor, but no attenuation was observed in vessels treated with GRK2 inhibitor. B: representative myograph tracing showing repetitive blue light (blue arrows) response in rat PAs in the absence of GRK2 inhibitor followed by light response in the presence of GRK2 inhibitor. C: proximity ligation assay (PLA) of human pulmonary arterial smooth muscle cells (hPASMCs) tested for GRK2 and Opn3 or Opn4 proximity. Control stain was performed with only the GRK2 antibody (n = 5). D: PLA of hPASMCs tested for phosphoserine and Opn3 or Opn4 proximity. Control stain was performed with only the phosphoserine antibody. (n = 5). ***P < 0.001.

Article Snippet: Human PASMCs (hPASMCs; PromoCell, Heidelberg, Germany) were maintained in smooth muscle cell growth medium 2 (PromoCell) in a humidified incubator at 37°C and 5% CO 2 and were used for experiments between passages 3 and 7 .

Techniques: Proximity Ligation Assay, Staining

Journal: bioRxiv

Article Title: Endothelial cell senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling

doi: 10.1101/2021.02.02.429321

Figure Lengend Snippet: ( A ) Real-time qPCR analysis for CDKIs and SASP factors in pulmonary artery ECs (PAECs) transfected with either GFP (control cells) or TRF2DN (premature senescent cells) (n = 5-8 each). ( B ) Schemes for co-culture experiments of PAECs and pulmonary artery SMCs (PASMCs). ( C ) Immunocytochemistry for Ki-67 in PASMCs directly (n = 7 each) or indirectly (n = 5 each) co-cultured with control or premature senescent PAECs. ( D ) Migration capacity was assessed by a modified Boyden chamber assay in PASMCs directly or indirectly co-cultured with control or premature senescent PAECs (n = 3 each). ( E ) Immunoblotting for cleaved caspase-3, total caspase-3, and GAPDH in PASMCs directly or indirectly co-culture PASMCs with control or premature senescent PAECs. Apoptosis was induced by incubating with 500 nM hydrogen peroxide for 3 h (n = 3 each). Data are presented as mean ± SEM. Two-tailed student’s t -test was used for the analysis of the differences between two groups. Two-way ANOVA with Tukey’s post hoc test was used for the analysis of the differences between groups more than three. Scale bars: 50 μM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: Human pulmonary arterial endothelial cells (PAECs) and smooth muscle cells (PASMCs) were purchased from PromoCell.

Techniques: Transfection, Co-Culture Assay, Immunocytochemistry, Cell Culture, Migration, Modification, Boyden Chamber Assay, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Endothelial cell senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling

doi: 10.1101/2021.02.02.429321

Figure Lengend Snippet: ( A ) Real-time qPCR analysis for differentiation markers in PASMCs directly (n = 4 each) or indirect (n=4 each) co-cultured with PAECs. ( B ) Phalloidin staining in PASMCs directly or indirectly co-cultured with PAECs. ( C ) Real-time qPCR analysis for Notch ligands in PAECs transfected with either GFP (n = 6 each) or TRF2DN (n = 6 each). Cells were treated with with either vehicle or 10 μM 5-azacytidine. Data are presented as mean ± SEM. Two-tailed student’s t -test was used for the analysis of the differences between two groups. Two-way ANOVA with Tukey’s post hoc test was used for the analysis of the differences between groups more than three. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: Human pulmonary arterial endothelial cells (PAECs) and smooth muscle cells (PASMCs) were purchased from PromoCell.

Techniques: Cell Culture, Staining, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: Endothelial cell senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling

doi: 10.1101/2021.02.02.429321

Figure Lengend Snippet: ( A ) Real-time qPCR analysis for Notch ligands in PAECs transfected with either GFP (control cells) or TRF2DN (premature senescent cells) (n= 6-8 each). ( B ) Real-time qPCR for Notch target genes in PASMCs directly or indirectly co-cultured with control or premature senescent PAECs (n = 4-5 each). ( C ) Immunocytochemistry for Ki-67 in PASMCs directly co-cultured with control or premature senescent PAECs. Cells were treated with either vehicle or 10 μM DAPT (n = 7-8 each). ( D ) Migration capacity was assessed by a modified Boyden chamber assay in PASMCs directly co-cultured with control or premature senescent PAECs. Cells were treated with either vehicle or 10 μM DAPT (n= 3 each). ( E ) Real-time qPCR analysis for Notch ligands in ECs isolated from the lungs of WT (n = 8-11) and TRF2DN-Tg (n = 9-10) mice exposed to chronic hypoxia. ( F ) Real-time qPCR analysis for Notch target genes in the lungs of WT (n = 11-12) and TRF2DN-Tg (n = 9-10) mice exposed to chronic hypoxia. Data are presented as mean ± SEM. Two-tailed student’s t -test was used for the analysis of the differences between two groups. Two-way ANOVA with Tukey’s post hoc test was used for the analysis of the differences between groups more than three. Scale bars: 50 μM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 .

Article Snippet: Human pulmonary arterial endothelial cells (PAECs) and smooth muscle cells (PASMCs) were purchased from PromoCell.

Techniques: Transfection, Cell Culture, Immunocytochemistry, Migration, Modification, Boyden Chamber Assay, Isolation, Two Tailed Test