Journal: Bio-protocol

Article Title: QsRNA-seq: A protocol for generating libraries for high-throughput sequencing of small RNAs

doi: 10.21769/BioProtoc.3179

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Article Snippet: RNase-free plastic pipette tips with filter (10 μl, 20 μl, 200 μl, 1,000 μl) 1.7 ml RNase-free microtubes 0.2 ml RNase-free PCR tubes miRVana miRNA Isolation Kit (Thermo Fisher scientific, catalog number: AM1560) or TRIzol reagent (Ambion, catalog number: 15596026) with Direct-Zol RNA MiniPrep Plus (Zymo Research, catalog number: R2072) RiboLock RNase Inhibitor (Thermo Fisher Scientific, catalog number: EO0381) T4 RNA ligase 1 (ssRNA Ligase) (NEB, catalog number: M0204S) T4 RNA ligase 2, truncated (NEB, catalog number: M0242S) RppH enzyme (NEB, catalog number M0356S) QScript Flex cDNA synthesis kit (Quanta, catalog number: 95049-025) Phusion High-Fidelity DNA Polymerase (NEB, catalog number: M0530S) Agencourt Ampure XP (Beckman Coulter, catalog number: A63881) or SPRI select reagent Kit (Beckman Coulter, catalog number: {"type":"entrez-nucleotide","attrs":{"text":"B23319","term_id":"2508950","term_text":"B23319"}} B23319 ) RNA BR assay kit (Quibit, catalog number: {"type":"entrez-protein","attrs":{"text":"Q10210","term_id":"1723278","term_text":"Q10210"}} Q10210 ) dsDNA HS assay kit (Quibit, catalog number: {"type":"entrez-protein","attrs":{"text":"Q10210","term_id":"1723278","term_text":"Q10210"}} Q10210 ) PEG 8000 (NEB, catalog number: B1004) Nuclease-free water (Sigma-Aldrich, catalog number: W4502) Ethyl Alcohol absolute Isopropyl-alcohol (Isopropanol) chemical grade Ethidium Bromide (hylabs, catalog number: BP451) Gel loading dye purple (6x) (NEB, catalog number: B7024) [Optional] Low-range ultra agarose (Bio-Rad, catalog number: 161-3107) ATP (NEB, catalog number: P0756S) DMSO (NEB, catalog number B0515) Tris (Spectrum, catalog number: s1519) Boric Acid (Bio-Lab, catalog number: 000201059100) EDTA (J.T.

Techniques:

Journal: Frontiers in Cardiovascular Medicine

Article Title: Developmental Angiogenesis Requires the Mitochondrial Phenylalanyl-tRNA Synthetase

doi: 10.3389/fcvm.2021.724846

Figure Lengend Snippet: The deficiency of FARS2 impairs cell motility, proliferation, invasion, and tube formation in human umbilical vein endothelial cells (HUVECs). (A) Representative images of scratch-wound assays of HUVECs 0 and 6 h at 48 h after transfection with a control (siCtrl) or FARS2 -specific (si- FARS2 ) siRNA. Scale bar = 200 μm. (B) Quantification of the healed wound area from (A) . Data are prepresented as the mean and SEM ( n = 10). *** P < 0.001 via ANOVA. (C) A CCK8-based cell proliferation assay of HUVECs at the indicated time-points after transfection with siCtrl or si- FARS2 . The measurements were made in triplicate (mean and SEM), and the results are indicative of three independent experiments. **** P < 0.0001. (D) Representative images of transwell-based migration assays of HUVECs 48 h after transfection with siCtrl or si- FARS2 . Scale bar = 200 μm. (E) Quantification of the number of migrated cells from (D) . *** P < 0.001. (F) Representative images of tube network assays of HUVECs 48 h after transfection with siCtrl or si- FARS2 . Scale bar = 200 μm. (G,H) Quantification of the branching points (G) and tube lengths (H) from (F) . The measurements were made in triplicate (mean and SEM), and the results are indicative of three independent experiments. **** P < 0.0001.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, Sciencell cat. # 8000) were used from passages 3–9 and cultured in endothelial cell medium (ECM, Sciencell cat. # 1001) containing 500 ml of basal medium, 5% fetal bovine serum (FBS, Sciencell cat. #0025), 1% endothelial cell growth supplement (Sciencell cat. #1052), and 1% antibiotic solution (P/S, Sciencell cat. #0503) in 5% CO 2 at 37°C.

Techniques: Transfection, Control, Proliferation Assay, Migration

Journal: Frontiers in Cardiovascular Medicine

Article Title: Developmental Angiogenesis Requires the Mitochondrial Phenylalanyl-tRNA Synthetase

doi: 10.3389/fcvm.2021.724846

Figure Lengend Snippet: FARS2 silencing causes mitochondrial dysfunction in human umbilical vein endothelial cells (HUVECs). (A) The oxygen consumption rate (OCR) in HUVECs transfected with siCtrl or si- FARS2 . The HUVECs were seeded 48 h after transfection with siRNAs and 12 h before analysis using a Seahorse XF24 analyzer. The OCR was measured continuously throughout the experimental period, both at baseline and in the presence of the indicated drugs. (B) Non-mitochondrial respiration, basal respiration, maximal respiration, proton leak, ATP production, and spare respiratory capacity in control and FARS2-deficient HUVECs. The measurements were made in triplicate (mean and SEM). ** P < 0.01, *** P < 0.001, **** P < 0.0001. (C) The effects of FARS2 knock-down on intracellular reactive oxygen species production by HUVECs. The measurements were made in triplicate (mean and SEM). * P < 0.05, (D) Quantification of total ATP levels in HUVECs 48 h after transfection with the indicated siRNAs. The measurements were made in triplicate (mean and SEM). ** P < 0.01.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, Sciencell cat. # 8000) were used from passages 3–9 and cultured in endothelial cell medium (ECM, Sciencell cat. # 1001) containing 500 ml of basal medium, 5% fetal bovine serum (FBS, Sciencell cat. #0025), 1% endothelial cell growth supplement (Sciencell cat. #1052), and 1% antibiotic solution (P/S, Sciencell cat. #0503) in 5% CO 2 at 37°C.

Techniques: Transfection, Control, Knockdown

Journal: Frontiers in Cardiovascular Medicine

Article Title: Developmental Angiogenesis Requires the Mitochondrial Phenylalanyl-tRNA Synthetase

doi: 10.3389/fcvm.2021.724846

Figure Lengend Snippet: The deficiency of FARS2 impairs angiogenesis by disrupting the Notch and Wnt signaling pathways. (A) The expression levels of genes involved in the Notch/Wnt pathways in control and fars2 zebrafish morphants, as determined by qRT-PCR analyses ( n = 6–10 individual embryos). *** P < 0.001, ** P < 0.01, * P < 0.05; ns, not significant. (B) The relative mRNA expression levels of Notch/Wnt pathway-related genes. The human umbilical vein endothelial cells (HUVECs) were transfected with the indicated siRNAs for 48 h and then harvested for qRT-PCR analysis. The measurements were made in triplicate (mean and SEM), and the results are indicative of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (C) Western blot analyses of NOTCH1 and β-catenin protein levels. The HUVECs were transfected with the indicated siRNAs for 48 h prior to analysis. (D) Quantification of the western blotting data described in (C) . The measurements were made in triplicate (mean and SEM). * P < 0.05, ** P < 0.01.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, Sciencell cat. # 8000) were used from passages 3–9 and cultured in endothelial cell medium (ECM, Sciencell cat. # 1001) containing 500 ml of basal medium, 5% fetal bovine serum (FBS, Sciencell cat. #0025), 1% endothelial cell growth supplement (Sciencell cat. #1052), and 1% antibiotic solution (P/S, Sciencell cat. #0503) in 5% CO 2 at 37°C.

Techniques: Protein-Protein interactions, Expressing, Control, Quantitative RT-PCR, Transfection, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Developmental Angiogenesis Requires the Mitochondrial Phenylalanyl-tRNA Synthetase

doi: 10.3389/fcvm.2021.724846

Figure Lengend Snippet: Developmental angiogenesis requires the mitochondrial phenylalanyl-tRNA synthetase. An overview of the mechanisms by which the deficiency of mitochondrial phenylalanyl-tRNA synthetase impairs angiogenesis by disrupting the Notch/Wnt pathways in zebrafish and human umbilical vein endothelial cells.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, Sciencell cat. # 8000) were used from passages 3–9 and cultured in endothelial cell medium (ECM, Sciencell cat. # 1001) containing 500 ml of basal medium, 5% fetal bovine serum (FBS, Sciencell cat. #0025), 1% endothelial cell growth supplement (Sciencell cat. #1052), and 1% antibiotic solution (P/S, Sciencell cat. #0503) in 5% CO 2 at 37°C.

Techniques: