Journal: Journal of the American Heart Association

Article Title: Monocrotaline Induces Endothelial Injury and Pulmonary Hypertension by Targeting the Extracellular Calcium–Sensing Receptor

doi: 10.1161/jaha.116.004865

Figure Lengend Snippet: Figure 2. MCT binding to CaSR in protein preparation. A, The proton spectrum of 5 lmol/L CaSR ECD and 1 mmol/L MCT in 10 mmol/L PBS containing 10% D2O (pH 7.5) obtained on NMR at the saturation of water (WL) and the enormous enhancement of proton signals in several down fields of 1 mmol/L MCT spectrum in the concomitant presence of 5 lmol/L CaSR ECD as well as the dose dependence of MCT. B, The proton spectrum of 5 lmol/L CaSR ECD and 1 mmol/L MCT in 10 mmol/L Tris in D2O (pD 7.8) obtained on NMR at the saturation of MCT and CaSR ECD, respectively, and the STD between MCT and CaSR ECD when irradiated on NMR for 3 seconds at 2.0 and 30 ppm for saturation and nonsaturation, respectively, of CaSR ECD. CaSR indicates extracellular calcium–sensing receptor; ECD, extracellular domain; MCT, monocrotaline; NMR, nuclear magnetic resonance; Ref, reference; STD, saturation transfer difference; WL, WaterLOGSY.

Article Snippet: After blocking with 5% BSA in a humidified chamber at room temperature overnight and draining off the blocking buffer, the PAECs were incubated with primary monoclonal antibody against CaSR (1:100 diluted in 0.5% BSA in PBS; ABclonal) in a humidified chamber at 4°C overnight.

Techniques: Binding Assay, Irradiation, Nuclear Magnetic Resonance

Journal: Journal of the American Heart Association

Article Title: Monocrotaline Induces Endothelial Injury and Pulmonary Hypertension by Targeting the Extracellular Calcium–Sensing Receptor

doi: 10.1161/jaha.116.004865

Figure Lengend Snippet: Figure 3. MCT binding to CaSR in cultured pulmonary endothelial cells and pulmonary arteries in vivo. A through C, Immunocytochemical stainings of CaSR (red) and nucleus (blue) in PAECs after incubation with and washout of the MCT labeled by FITC (FITC-MCT; green in A and C) or FITC alone (B). The confocal images represented the similar results of 3 separate experiments examining a total of 71 to 96 individual PAECs for each. D, Representative immunoblotting analysis of the protein lysates of pulmonary arteries isolated from blank control, DMSO, Calhex 231 or MCT-administered rats and heated at a series of temperatures from 42 to 72°C for 3 minutes. E, Statistical regression (4 parameter logistic regressions, P<0.05) of separate immunoblots generating the cellular thermal shift assay curves for the enhanced thermal stability of CaSR by Calhex 231 and MCT. CaSR indicates extracellular calcium–sensing receptor; ECD, extracellular domain; FITC, fluorescein isothiocyanate; MCT, monocrotaline; N-FRET, net fluorescence resonance energy transfer; PAEC, pulmonary artery endothelial cell.

Article Snippet: After blocking with 5% BSA in a humidified chamber at room temperature overnight and draining off the blocking buffer, the PAECs were incubated with primary monoclonal antibody against CaSR (1:100 diluted in 0.5% BSA in PBS; ABclonal) in a humidified chamber at 4°C overnight.

Techniques: Binding Assay, Cell Culture, In Vivo, Incubation, Labeling, Western Blot, Isolation, Control, Thermal Shift Assay, Förster Resonance Energy Transfer

Journal: Journal of the American Heart Association

Article Title: Monocrotaline Induces Endothelial Injury and Pulmonary Hypertension by Targeting the Extracellular Calcium–Sensing Receptor

doi: 10.1161/jaha.116.004865

Figure Lengend Snippet: Figure 4. MCT-induced mobilization of [Ca2+]i and endothelial injury via CaSR. A through G, Representative curves of [Ca2+]i responses to PBS, 1 lmol/L Calhex 231, or 2.5 mmol/L MCT in PAECs in the absence or presence of 1 lmol/L Calhex 231 or to 2.5 mmol/L MCT in PAECs transfected with CaSR shRNA, control shRNA, or the vector. H, Statistical summary of [Ca2+]i baseline and MCT-induced peak responses. The sample size indicated is the total number of PAECs from 3 independent experiments for each (*P<0.001 vs control, #P<0.001 vs MCT). After 24-hour incubation with 2.5 mmol/L MCT in the absence or presence of 1 lmol/ L Calhex 231 in PAECs or in PAECs transfected with CaSR shRNA, control shRNA, or the vector, CCK-8 staining and Western blots were performed to quantify the cell survival rate and cleaved caspase 3, respectively. Summarized were 4 independent measurements of the cell survival rate normalized to control (I), and the representative Western blot and comparison of cleaved caspase 3 levels from 3 independent experiments (J), *P<0.05 vs control, #P<0.05 vs MCT. [Ca2+]i indicates cytosolic Ca2+; CaSR indicates extracellular calcium–sensing receptor; MCT, monocrotaline; PAEC, pulmonary artery endothelial cell; shRNA, short hairpin RNA.

Article Snippet: After blocking with 5% BSA in a humidified chamber at room temperature overnight and draining off the blocking buffer, the PAECs were incubated with primary monoclonal antibody against CaSR (1:100 diluted in 0.5% BSA in PBS; ABclonal) in a humidified chamber at 4°C overnight.

Techniques: Transfection, shRNA, Control, Plasmid Preparation, Incubation, CCK-8 Assay, Staining, Western Blot, Comparison

Journal: Journal of the American Heart Association

Article Title: Monocrotaline Induces Endothelial Injury and Pulmonary Hypertension by Targeting the Extracellular Calcium–Sensing Receptor

doi: 10.1161/jaha.116.004865

Figure Lengend Snippet: Figure 5. MCT-induced assembly of CaSR. A through C, Representative images of CFP, YFP, FRET, and Fc in PAECs coexpressed with CFP CaSR ECD and YFP CaSR ECD before and after exposure to vehicle (A) or 2.5 mmol/L monocrotaline (B) and the quantification of N- FRET (C). The sample size indicated is the number of individual PAECs examined, *P<0.05. D, Representative Western blot and normalized densitometry analysis. *#P<0.05 vs 55 to 72 or 240 to 300 kDa in control, respectively, n=3 separate experiments for each. CaSR indicates extracellular calcium–sensing receptor; CFP, cyan fluorescent protein; ECD, extracellular domain; Fc, corrected fluorescence resonance energy transfer; FRET, fluorescence resonance energy transfer; MCT, monocrotaline; N-FRET; net fluorescence resonance energy transfer; PAEC, pulmonary artery endothelial cell; YFP, yellow fluorescent protein.

Article Snippet: After blocking with 5% BSA in a humidified chamber at room temperature overnight and draining off the blocking buffer, the PAECs were incubated with primary monoclonal antibody against CaSR (1:100 diluted in 0.5% BSA in PBS; ABclonal) in a humidified chamber at 4°C overnight.

Techniques: Western Blot, Control, Förster Resonance Energy Transfer

Journal: Journal of the American Heart Association

Article Title: Monocrotaline Induces Endothelial Injury and Pulmonary Hypertension by Targeting the Extracellular Calcium–Sensing Receptor

doi: 10.1161/jaha.116.004865

Figure Lengend Snippet: Figure 6. Attenuation of MCT-induced pulmonary hypertension by CaSR inhibitor. Alterations of Ppa (A), PVR (B), Fulton index (the index of right ventricular hypertrophy, RV/[LV+S]) (C), representative hematoxylin and eosin stainings of a resistance pulmonary artery (D) and summary of pulmonary artery muscularization (E) in control rats or rats treated with MCT, Calhex 231, or MCT plus Calhex 231. *P<0.05 vs control, #P<0.05 vs MCT. The sample size indicated is the number of rats for each group. CaSR indicates extracellular calcium–sensing receptor; LV, left ventricle; MCT, monocrotaline; Ppa, pulmonary artery pressure; PVR, pulmonary vascular resistance; RV, right ventricle; S, septum.

Article Snippet: After blocking with 5% BSA in a humidified chamber at room temperature overnight and draining off the blocking buffer, the PAECs were incubated with primary monoclonal antibody against CaSR (1:100 diluted in 0.5% BSA in PBS; ABclonal) in a humidified chamber at 4°C overnight.

Techniques: Control

Journal: Journal of the American Heart Association

Article Title: Monocrotaline Induces Endothelial Injury and Pulmonary Hypertension by Targeting the Extracellular Calcium–Sensing Receptor

doi: 10.1161/jaha.116.004865

Figure Lengend Snippet: Figure 7. Attenuation of MCT-induced pulmonary hypertension by CaSR knockdown. A through E, Alterations of Ppa (A), PVR (B), Fulton index, RV/(LV+S) (C), representative HE stainings of a resistance pulmonary artery (D), and summary of pulmonary artery muscularization (E) in control rats and MCT-treated rats without or with intravenous pretransduction of control shRNA, vector, or CaSR shRNA, respectively. *P<0.05 vs control, #P<0.05 vs MCT. F through J, Alterations of Ppa (F), PVR (G), Fulton index, RV/(LV+S) (H), representative HE stainings of a resistance pulmonary artery (I), and summary of pulmonary artery muscularization (J) in control rats and MCT-treated rats without or with the intratracheal pretransduction of control shRNA, vector, or CaSR shRNA, respectively. *P<0.05 vs control, #P<0.05 vs MCT. The sample size indicated is the number of rats for each group. The groups of blank control and MCT treatment were included in Figures 6 through 8 for comparison. CaSR indicates extracellular calcium–sensing receptor; HE, hematoxylin and eosin; LV, left ventricle; MCT, monocrotaline; Ppa, pulmonary artery pressure; PVR, pulmonary vascular resistance; RV, right ventricle; S, septum; shRNA, short hairpin RNA.

Article Snippet: After blocking with 5% BSA in a humidified chamber at room temperature overnight and draining off the blocking buffer, the PAECs were incubated with primary monoclonal antibody against CaSR (1:100 diluted in 0.5% BSA in PBS; ABclonal) in a humidified chamber at 4°C overnight.

Techniques: Knockdown, Control, shRNA, Plasmid Preparation, Comparison

Journal: Journal of the American Heart Association

Article Title: Monocrotaline Induces Endothelial Injury and Pulmonary Hypertension by Targeting the Extracellular Calcium–Sensing Receptor

doi: 10.1161/jaha.116.004865

Figure Lengend Snippet: Figure 8. Attenuation of MCT-induced pulmonary hypertension by CaSR knockout. Alterations of Ppa (A), PVR (B), Fulton index, RV/(LV+S) (C), representative HE stainings of a resistance pulmonary artery (D), and summary of pulmonary artery muscularization (E) in control rats and MCT-treated PTH knockout (PTH/CaSR+/+) rats or PTH and CaSR double-knockout (PTH/CaSR/) rats, respectively. *P<0.05 vs control, #P<0.05 vs MCT. The number in the bar indicates the total rats examined for each group. CaSR indicates extracellular calcium–sensing receptor; HE, hematoxylin and eosin; LV, left ventricle; MCT, monocrotaline; Ppa, pulmonary artery pressure; PTH, parathyroid hormone; PVR, pulmonary vascular resistance; RV, right ventricle; S, septum; shRNA, short hairpin RNA; WT, wild type.

Article Snippet: After blocking with 5% BSA in a humidified chamber at room temperature overnight and draining off the blocking buffer, the PAECs were incubated with primary monoclonal antibody against CaSR (1:100 diluted in 0.5% BSA in PBS; ABclonal) in a humidified chamber at 4°C overnight.

Techniques: Knock-Out, Control, Double Knockout, shRNA