recombinant mouse endostatin  (R&D Systems)

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Functional Impact of Human Genetic Variants of COL18A1 /Endostatin on Pulmonary Endothelium

doi: 10.1165/rcmb.2019-0056OC

Figure Lengend Snippet: Endostatin exposure inhibits pulmonary endothelial cell (PEC) proliferation and migration and promotes PEC cell death. Human macrovascular pulmonary endothelial cells (HPAECs) (left) and human microvascular pulmonary endothelial cells (HLMVECs) (right). (A) PECs were treated with vehicle or recombinant endostatin (rES) (0.6 μg/ml or 1.2 μg/ml) for 4 hours, after which migration was assessed with a modified Boyden Transwell assay. Migration Index = normalized number of PECs relative to vehicle-treated arm. (B) PECs treated with vehicle or rES (0.6 μg/ml or 1.2 μg/ml) for 48 hours, after which proliferative antigen Ki67 was quantified using flow cytometry. Proliferative Index = percentage of Ki67-positive cells normalized to vehicle control. (C) PECs were exposed to rES for 18 hours, and the percentage of cells staining for active caspase 3 were assessed with flow cytometry. Apoptosis Index = percentage of caspase 3–positive cells normalized to vehicle control. n = 4 per treatment arm. *P ≤ 0.05 compared with vehicle. Data expressed as mean ± SEM.

Article Snippet: Reagents used included recombinant human endostatin (rES) and recombinant human TSP-1 (R&D Systems) and the proteasome inhibitor MG132 (Cell Signaling Technology).

Techniques: Migration, Recombinant, Modification, Transwell Assay, Flow Cytometry, Control, Staining

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Functional Impact of Human Genetic Variants of COL18A1 /Endostatin on Pulmonary Endothelium

doi: 10.1165/rcmb.2019-0056OC

Figure Lengend Snippet: Endostatin suppresses ID1 protein expression through a post-transcriptional proteasome-dependent mechanism, and ID1 is necessary for rES-induced angiostasis. (A) PECs were exposed to rES for an increasing length of time (15–90 min), and total cellular ID1 protein expression was evaluated by Western blot analysis (WB). WB is representative of several independent experiments. The total number per treatment arm included in the analysis was between four and nine samples. (B) PECs were exposed to rES (1.2 μg/ml) or vehicle for 30 minutes. RNA was extracted, and ID1 mRNA expression was assessed relative to ACTB/actin expression with qRT-PCR. (C) PECs were exposed to rES and the proteasome inhibitor MG132 (1 μM) or vehicle for 30 minutes. ID1 protein expression was evaluated with WB analysis. For WB, ID1 expression was quantified by protein densitometry, normalized to actin, and displayed as the proportion of ID1 expression in vehicle-treated cells. (D–F) PECs were transduced with adenovirus encoding ID1 (AD ID1) or null adenovirus (AD Null). (D) Cells were exposed to rES for 4 hours, and migration was assessed with a Transwell assay. (E) Cells were exposed to rES for 48 hours, and proliferation was assessed via flow cytometry. (F) Cells were exposed to rES for 18 hours, and caspase 3 activity was assessed with flow cytometry. For all experiments, data are displayed relative to vehicle treatment arm for each respective adenovirus treatment arm. n = 4–9 per treatment arm. *P ≤ 0.05 compared with vehicle. Data expressed as mean ± SEM. R.U. = relative units.

Article Snippet: Reagents used included recombinant human endostatin (rES) and recombinant human TSP-1 (R&D Systems) and the proteasome inhibitor MG132 (Cell Signaling Technology).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transduction, Migration, Transwell Assay, Flow Cytometry, Activity Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Functional Impact of Human Genetic Variants of COL18A1 /Endostatin on Pulmonary Endothelium

doi: 10.1165/rcmb.2019-0056OC

Figure Lengend Snippet: Endostatin exposure results in increased THBS1/TSP-1 expression. (A) HPAECs were exposed to rES or vehicle for increasing durations (0.5–8 h). RNA was extracted, and THBS1 mRNA expression was assessed relative to ACTB/actin expression with qRT-PCR. (B) HLMVECs were exposed to rES for 4 hours, mRNA was extracted, and THBS1 mRNA expression was assessed relative to ACTB/actin via qRT-PCR. (C) PECs were treated with the proteasome inhibitor MG132 (1 μM) or its carrier and then challenged with rES or vehicle for 4 hours. RNA was extracted, and THBS1 mRNA expression was assessed relative to ACTB/actin expression with qRT-PCR. Data are displayed as the proportion of THBS1 mRNA expression in vehicle-treated cells. (D and E) PECs were exposed to rES for increasing durations (15 min to 24 h). Cellular supernatants (extracellular) and lysates (cell associated) were collected, and protein expression was evaluated via WB analysis. (D) WB of cell-associated protein expression of TSP-1 and quantified by protein densitometry. Cell-associated TSP-1 normalized to actin and displayed as the proportion of TSP-1 protein expression in vehicle-treated cells. (E) WB of extracellular protein expression of TSP-1 quantified by densitometry. Extracellular TSP-1 expression displayed as the proportion of TSP-1 expression in vehicle-treated cells. n = 3–6 per treatment arm. *P ≤ 0.05 compared with vehicle. Data are expressed as mean ± SEM. TSP-1 = thrombospondin 1.

Article Snippet: Reagents used included recombinant human endostatin (rES) and recombinant human TSP-1 (R&D Systems) and the proteasome inhibitor MG132 (Cell Signaling Technology).

Techniques: Expressing, Quantitative RT-PCR