normal human paecs  (Lonza)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Endothelial Dec1-PPARγ Axis Impairs Proliferation and Apoptosis Homeostasis Under Hypoxia in Pulmonary Arterial Hypertension

doi: 10.3389/fcell.2021.757168

Figure Lengend Snippet: Dec1 is upregulated and PPARγ is downregulated in PAH. (A) Expression levels of Dec1 and PPARγ were determined by western blot in pulmonary arteries from PAH ( n = 4) and healthy control patients ( n = 4). (B) Human pulmonary arterial endothelial cells (PAECs) were isolated from PAH and healthy control patients ( n = 4 per group), and western blot was performed to determine expressions of the Dec1–PPARγ axis. (C–F) Human normal PAECs (hnPAECs) were treated with serum deprivation (C) , 100 μM H 2 O 2 (D) , 20 ng/ml TGF-β1 (E) , and 50 ng/ml IL-6 (F) in vitro . Expression levels of Dec1 and PPARγ in PAECs were determined by western blot (left part) at the indicated times. One-way ANOVA test was applied for statistical analysis (right part). Data are represented as mean ± SEM. Experiments were repeated for three independent times. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Human normal PAECs (hnPAECs) were purchased from Lonza (Basel, Switzerland) and cultured for three to six passages prior to use in the medium (EBM-2) provided by the manufacturers.

Techniques: Expressing, Western Blot, Control, Isolation, In Vitro

Journal: Frontiers in Cell and Developmental Biology

Article Title: Endothelial Dec1-PPARγ Axis Impairs Proliferation and Apoptosis Homeostasis Under Hypoxia in Pulmonary Arterial Hypertension

doi: 10.3389/fcell.2021.757168

Figure Lengend Snippet: Effect of Dec1 on PAECs survival under hypoxia. (A,B) Human normal PAECs (hnPAECs) were treated with 3% O 2 for 0, 24, 48, and 96 h and then performed real-time polymerase chain reaction (RT-PCR) or Western blot to determine the mRNA and protein levels of the target genes, respectively. (A) mRNA expression levels of Dec1. (B) Protein expression levels of Dec1 and PPARγ. One-way ANOVA test was applied for statistical analysis. (C–F) After 12 h of transient transfection, cells were treated with 3% O 2 for 6, 12, 24, and 48 h and monitored the cell viability vs. time by the cell counting kit 8 (CCK-8) assay. Cells were plotted cell viability at the indicated times. (C) Healthy- and PAH–PAECs with no transfections. (D) PAH–PAECs transfected with Dec1-shRNA (Sh-Dec1) or the scrambled RNA (Sh-Ctrl) plasmids. (E) hnPAECs transfected with Dec1 overexpression (Ov-Dec1) or the empty control (Ov-Ctrl) plasmids. (F) hnPAECs transfected with Dec1–shRNA (Sh-Dec1) or the scrambled RNA (Sh-Ctrl) plasmids. Two-way ANOVA test was applied for statistical analysis. Data are represented as mean ± SEM. Experiments were repeated for three independent times. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Human normal PAECs (hnPAECs) were purchased from Lonza (Basel, Switzerland) and cultured for three to six passages prior to use in the medium (EBM-2) provided by the manufacturers.

Techniques: Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Cell Counting, CCK-8 Assay, shRNA, Over Expression, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Endothelial Dec1-PPARγ Axis Impairs Proliferation and Apoptosis Homeostasis Under Hypoxia in Pulmonary Arterial Hypertension

doi: 10.3389/fcell.2021.757168

Figure Lengend Snippet: Effect of hypoxia on phenotypes of PAECs. Isolated healthy- or PAH–PAECs were treated with hypoxia (3% O 2 ) for 24 h. Human normal PAECs (hnPAECs) were preconditioned with normoxia (21% O 2 , pNor-PAECs) or hypoxia (3% O 2 , pHyp-PAECs) for 1 week. Then these cells were assessed proliferation and apoptosis by immunofluorescence staining of Ki-67 and flow cytometry under hypoxia (3% O 2 ), respectively. In proliferation assay, Ki-67 positive cells (red) with nuclear DAPI counterstain (blue) indicated the proliferating cells. In apoptosis map, cells were divided into four subpopulations: necrotic cells (Q1: annexin V FITC–/PI+), late apoptotic cells (Q2: annexin V FITC+/PI+), live cells (Q3: annexin V FITC–/PI–), and early apoptotic cells (Q4: annexin V FITC+/PI–). The proliferative (Cyclin B1 and Cyclin D1) and apoptotic factors (Bax, Bcl-2, and cleaved-caspase 3) were detected by Western blot. (A) Immunofluorescence staining of Ki-67 (left part, scale bars = 50 μm) and the proliferation rate (right part). (B) Expressions of Cyclin B1 and Cyclin D1. (C) Flow cytometry (left part) and the apoptosis rate (right part). (D) Expressions of Bax, Bcl-2, and cleaved caspase 3. (E) Immunofluorescence staining of Ki-67 (left part, scale bars = 50 μm) and the proliferation rate (right part). (F) Expressions of Cyclin B1 and Cyclin D1. (G) Flow cytometry (left part) and the apoptosis rate (right part). (H) Expressions of Bax, Bcl-2, and cleaved caspase 3. One-way ANOVA test was applied for statistical analysis. Data are represented as mean ± SEM. Experiments were repeated for three independent times. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Human normal PAECs (hnPAECs) were purchased from Lonza (Basel, Switzerland) and cultured for three to six passages prior to use in the medium (EBM-2) provided by the manufacturers.

Techniques: Isolation, Immunofluorescence, Staining, Flow Cytometry, Proliferation Assay, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Endothelial Dec1-PPARγ Axis Impairs Proliferation and Apoptosis Homeostasis Under Hypoxia in Pulmonary Arterial Hypertension

doi: 10.3389/fcell.2021.757168

Figure Lengend Snippet: Hypoxia promotes the PAH phenotypes of PAECs by induction of Dec1. Human normal PAECs (hnPAECs) were transfected with Dec1–shRNA (Sh-Dec1) or scrambled RNA (Sh-Ctrl) and then preconditioned with normoxia (21% O 2 , pNor-PAECs) or hypoxia (3% O 2 , pHyp-PAECs) for 1 week. Next, these cells were assessed proliferation and apoptosis by immunofluorescence staining of Ki-67 and flow cytometry under hypoxia (3% O 2 ), respectively. In proliferation assay, Ki-67-positive cells (red) with nuclear DAPI counterstain (blue) indicated the proliferating cells. In apoptosis map, cells were divided into four subpopulations: necrotic cells (Q1: annexin V FITC–/PI+), late apoptotic cells (Q2: annexin V FITC+/PI+), live cells (Q3: annexin V FITC–/PI–), and early apoptotic cells (Q4: annexin V FITC+/PI–). The proliferative (Cyclin B1 and Cyclin D1) and apoptotic factors (Bax, Bcl-2 and cleaved-caspase 3) were also detected by western blot. (A) Immunofluorescence staining of Ki-67 (left part, scale bars = 50 μm) and the proliferation rate (right part). (B,C) The expressions of Cyclin B1 (B) and Cyclin D1 (C) . (D) Flow cytometry (left part) and the apoptosis rate (right part). (E,F) The expressions of Bax, Bcl-2 (E) , and cleaved caspase 3 (F) . One-way ANOVA test was applied for statistical analysis. Data are represented as mean ± SEM. Experiments were repeated for three independent times. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Human normal PAECs (hnPAECs) were purchased from Lonza (Basel, Switzerland) and cultured for three to six passages prior to use in the medium (EBM-2) provided by the manufacturers.

Techniques: Transfection, shRNA, Immunofluorescence, Staining, Flow Cytometry, Proliferation Assay, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Endothelial Dec1-PPARγ Axis Impairs Proliferation and Apoptosis Homeostasis Under Hypoxia in Pulmonary Arterial Hypertension

doi: 10.3389/fcell.2021.757168

Figure Lengend Snippet: Dec1 promotes the PAH phenotypes of PAECs by inhibition of PPARγ. (A) Human normal PAECs (hnPAECs) were transfected with Dec1 Sh-RNA (Sh-Dec1) or the scrambled control (Sh-Ctrl) plasmids and then treated with normoxia (21% O 2 ) or hypoxia (3% O 2 ) for 24 h. Protein levels of Dec1 and PPARγ were detected by Western blot in the indicated cells. (B) hnPAECs were transfected with Dec1 overexpression (Ov-Dec1) or the empty control (Ov-Ctrl) plasmids and treated with normoxia (21% O 2 ) for 24 h. Protein levels of Dec1 and PPARγ were detected by western blot in the indicated cells. (C,D) Human normal PAECs (hnPAECs) were transfected with Ov-Ctrl, Ov-Dec1, Ov-Dec1 + Ov-Ctrl, and Ov-Dec1 + Ov-PPARγ plasmids and treated with normoxia (21% O 2 ) for 24 h. Then these cells were assessed proliferation and apoptosis by immunofluorescence staining of Ki-67 ( C , scale bars = 50 μm) and flow cytometry (D) , respectively. In proliferation assay, Ki-67-positive cells (red) with nuclear DAPI counterstain (blue) indicated the proliferating cells. In apoptosis map, cells were divided into four subpopulations: necrotic cells (Q1: annexin V FITC–/PI+), late apoptotic cells (Q2: annexin V FITC+/PI+), live cells (Q3: annexin V FITC–/PI–), and early apoptotic cells (Q4: annexin V FITC+/PI–). One-way ANOVA test was applied for statistical analysis. Data are represented as mean ± SEM. Experiments were repeated for three independent times. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Human normal PAECs (hnPAECs) were purchased from Lonza (Basel, Switzerland) and cultured for three to six passages prior to use in the medium (EBM-2) provided by the manufacturers.

Techniques: Inhibition, Transfection, Control, Western Blot, Over Expression, Immunofluorescence, Staining, Flow Cytometry, Proliferation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Endothelial Dec1-PPARγ Axis Impairs Proliferation and Apoptosis Homeostasis Under Hypoxia in Pulmonary Arterial Hypertension

doi: 10.3389/fcell.2021.757168

Figure Lengend Snippet: Schema of the signaling pathways leading to disruption of Dec1-PPARγ axis in PAECs in response to hypoxia.

Article Snippet: Human normal PAECs (hnPAECs) were purchased from Lonza (Basel, Switzerland) and cultured for three to six passages prior to use in the medium (EBM-2) provided by the manufacturers.

Techniques: Protein-Protein interactions, Disruption