Journal: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet: Characterization of an AAV6.2FF-hACE2 transduction model for SARS-CoV-2 infection of wild-type mice (A) Diagram of AAV genome expressing hACE2 from the CASI promoter. (B) Western blot of HEK293 cells transduced with AAV6.2FF-hACE and probed with an anti-hACE2 antibody. (C) BALB/c mice were administered 1 x 10 11 vg of AAV-Luc intranasally and imaged 10 days later using an IVIS imager. (D–F) (D) IFA images of lungs harvested from BALB/c mice infected intranasally with 1 x 10 11 vg of AAV-hACE2 or AAV-Luc and euthanized 10 days later. Lungs were stained with a rabbit anit-hACE2 antibody and imaged at 20 X (scale bar, 50mM). Viral RNA (E), and virus TCID50 titers (F) were determined in respiratory tissues on days 2 and 4 post-infection. n = 6 (3M, 3F). Statistical significance determined by Mann-Whitney test. ∗ = p < 0.05, ∗∗ = p < 0.01.

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: Transduction, Infection, Expressing, Western Blot, Staining, MANN-WHITNEY

Journal: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet: SARS-CoV-2 spike DNA antigens protect from viral replication in vivo (A) Mice were immunized once or twice separated by four weeks with 10ug of pS via electroporation. Serum was collected at day 18 post-final immunization. At 35 days post-final immunization mice were infected intranasally with adeno-associated virus expressing human ACE2 (white). 17 days following AAV6-ACE2 transduction, animals were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2 VIDO-01 P2. Four days post infection, animals were sacrificed to quantify viral replication. SARS-CoV-2 specific serum IgG endpoint titers (B) and pseudoviral neutralization titers (C) at day 18 post-final immunization. Replication competent virus (D), and viral RNA (E) in the lungs four-days post-infection. Pearson correlations between virus titer and serum IgG endpoints (F) and neutralization titers (G). Pearson correlations between viral RNA copies and serum IgG endpoints (H) and neutralization titers (I). Each point represents the average of duplicate samples from an individual animal, bars represent the mean, lines represent the median, and error bars represent the SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by student's t-test (A and B), or Kruskall-Wallis ANOVA (D and E). Spearman correlations were used to determine relationships (F-I). Data are representative of one experiment with n = 5 males (squares) and 5 females (circles) per group.

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: In Vivo, Electroporation, Infection, Expressing, Transduction, Neutralization

Journal: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet:

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: Recombinant, Plasmid Preparation, Enzyme-linked Immunospot, Software

Journal: Journal of Molecular and Cellular Cardiology

Article Title: Angiotensin peptide synthesis and cyclic nucleotide modulation in sympathetic stellate ganglia

doi: 10.1016/j.yjmcc.2019.11.157

Figure Lengend Snippet: Angiotensin synthesizing enzymes and precursors are expressed in human stellate ganglia. In human stellate ganglia the presence of the mRNA transcripts encoding Agt ( n = 4), Ren ( n = 3) , Ace (n = 4) , Ace2 (n = 3) , Agtr1 (n = 4) , Agtr2 (n = 3) and Mas1 (n = 4) were confirmed by qRT-PCR. The qRT-PCR raw counts for the genes of interest were normalized to the control gene B2m using the ∆C T method and expressed as ∆C T mean ± SEM (a). ELISAs were used to demonstrate the protein expression of the relevant proteins of interest including Agt, Ren, AngII, ACE2 and Ang1–7 in human stellate ganglia. Agt was found to be highly expressed in human stellate ganglia ( n = 2, ~53,694 pg/mg), as was Ren (n = 3, 2005 ± 388 pg/mg). AngII (n = 3, 188.7 ± 15.37 pg/mg), ACE2 (n = 2, 171.9 ± 2.60 pg/mg) and Ang1–7 (n = 3, 179.9 ± 6.13 pg/mg) were also identified and were found to have similar levels of expression (b). Data are displayed as mean ± SEM. A model diagram depicts AngII and Ang1–7 release from the stellate ganglia and the proposed pre-and post-synaptic effects.

Article Snippet: Enzyme immuno assays (EIA) or enzyme-linked immunosorbent assays (ELISA) were conducted to detect the presence of the following proteins of interest in human stellate ganglia: AGT (CSB-E08564h, Cusabio), renin (dren00, R&DSystems), AngII (RAB0010-1KT, Sigma), ACE2 (LS-F5886, LSBio), Ang1-7 (CSB-E14242h).

Techniques: Quantitative RT-PCR, Control, Expressing

Journal: Journal of Molecular and Cellular Cardiology

Article Title: Angiotensin peptide synthesis and cyclic nucleotide modulation in sympathetic stellate ganglia

doi: 10.1016/j.yjmcc.2019.11.157

Figure Lengend Snippet: Transcripts of angiotensin synthesizing genes were observed in the rat sympathetic stellate ganglia in the RNA-seq dataset. The transcriptome of the sympathetic stellate ganglia was sequenced using stellate ganglia extracted from four-week-old male Wistar rats ( n = 5) and age-matched male prehypertensive SHR (preSHR, n = 5). A KEGG analysis was carried out using the differentially expressed transcripts where the gene input was selected using the Benjamini-Hochburg p.adj < 0.05. The KEGG group ‘Renin Secretion’ was found to be significantly altered in the preSHR ganglia, where the gene input was selected using the Benjamini-Hochburg p.adj < 0.05 (a). A full list of the genes, the fold changes and respective levels of significance are reported in . The AngII and Ang1–7 synthesis pathways are outlined (b). Transcripts encoding the enzymes and precursors classically involved in the synthesis of AngII and Ang1–7 were identified in young rat stellate ganglia (b), where the relevant transcripts included Angiotensinogen ( Agt), Renin ( Ren ) and the Angiotensin Converting Enzymes ( Ace, Ace2 ). The transcripts for AngII receptors type 1 and 2 ( Agtr1a, Agtr1b, Agtr2) and for the Ang1–7 receptor Mas ( M as1 ) were also observed (c). Transcript abundances were not found to be differentially expressed in preSHR vs. Wistar ganglia, with the exception of Agtr1a that was significantly downregulated in the preSHR stellate ganglia (p. adj = 3.72 × 10 −8 , Salmon-DESeq2 method [ , ]).

Article Snippet: Enzyme immuno assays (EIA) or enzyme-linked immunosorbent assays (ELISA) were conducted to detect the presence of the following proteins of interest in human stellate ganglia: AGT (CSB-E08564h, Cusabio), renin (dren00, R&DSystems), AngII (RAB0010-1KT, Sigma), ACE2 (LS-F5886, LSBio), Ang1-7 (CSB-E14242h).

Techniques: RNA Sequencing

Journal: Journal of Molecular and Cellular Cardiology

Article Title: Angiotensin peptide synthesis and cyclic nucleotide modulation in sympathetic stellate ganglia

doi: 10.1016/j.yjmcc.2019.11.157

Figure Lengend Snippet: Angiotensinergic mRNA transcript validation by qRT-PCR in rat stellate ganglia. The presence of the RNA transcripts Agt, Ren, Ace, Ace2, Agtr1a, Agtr1b, Agtr2 and Mas1 was confirmed by qRT-PCR in sympathetic stellate ganglia from four-week Wistar and preSHR ganglia (a), and 16-week adult Wistar and SHR (b). The qRT-PCR raw counts were first normalized to a control gene B2m as per the comparative (∆C T ) method . Each data point corresponds to one stellate RNA sample from one rat. Data are displayed as ∆C T mean ± SEM. FRET microscopy was conducted on sympathetic stellate neurons obtained from Wistar ( n = 11 rats, 3 cultures, 20 cells) and preSHR rats ( n = 9 rats, 3 cultures, 19 cells). Cells were transduced with the cGi500 FRET sensor and randomly selected for imaging. Increases in cGMP generation was observed in sympathetic neurons in response to Ang1–7 and AngII (c, d). Maximal FRET changes were evoked following administration of a combination of the NO-donor Sin-1 (10 μM) and the PDE inhibitor IBMX (100 μM). There was significantly greater cGMP generation in response to AngII in Wistar vs. preSHR neurons (two-way ANOVA, p = .0403). Peak FRET changes were obtained in response to AngII or Ang1–7 and converted to percentage FRET changes and values are depicted as a proportion of the maximal FRET change (%). There was no difference in peak FRET responses in response to Ang1–7 or between strains (d). Data are displayed as mean ± SEM.

Article Snippet: Enzyme immuno assays (EIA) or enzyme-linked immunosorbent assays (ELISA) were conducted to detect the presence of the following proteins of interest in human stellate ganglia: AGT (CSB-E08564h, Cusabio), renin (dren00, R&DSystems), AngII (RAB0010-1KT, Sigma), ACE2 (LS-F5886, LSBio), Ang1-7 (CSB-E14242h).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Control, Microscopy, Transduction, Imaging

Journal: Journal of Molecular and Cellular Cardiology

Article Title: Angiotensin peptide synthesis and cyclic nucleotide modulation in sympathetic stellate ganglia

doi: 10.1016/j.yjmcc.2019.11.157

Figure Lengend Snippet: Model diagram depicts angiotensin synthesis and pre- and post-synaptic signaling pathways. In sympathetic stellate neurons, the classical pathway for Angiotensin II (AngII) synthesis occurs by sequential enzymatic cleavage of Angiotensinogen (Agt) by renin and Angiotensin Converting Enzymes (ACE). AngII is hydrolyzed by ACE2 producing the bioactive metabolite of Angiotensin 1–7 (Ang1–7). We identified the presence of precursors and transcripts encoding these enzymes and depict here a proposed model for angiotensin synthesis (a). We also identified the presence of AngII and Ang1–7 receptors on sympathetic stellate ganglia of human and rat. AngII has been shown to elevate intracellular Ca 2+ and enhance noradrenaline release via actions at AT 1 R [ , ]. Conversely Ang1–7-dependent activation of its cognate receptor, Mas R, has been shown to couple to NO in the brain and several other receptor sites . In this study, we show that administration of both AngII and Ang1–7 elevate cGMP in the rat stellate ganglia. We and others have previously demonstrated the importance of NO-cGMP signaling in reducing [Ca 2+ ] i [ , ] and end-organ transmission in peripheral sympathetic stellate nerves [ , , , ] although the effects of Ang1–7 may be biphasic . Dotted lines indicate intermediates in these intracellular signaling pathway. Several effects of AngII and Ang1–7 on the myocardium have been established [ , , , , , ].

Article Snippet: Enzyme immuno assays (EIA) or enzyme-linked immunosorbent assays (ELISA) were conducted to detect the presence of the following proteins of interest in human stellate ganglia: AGT (CSB-E08564h, Cusabio), renin (dren00, R&DSystems), AngII (RAB0010-1KT, Sigma), ACE2 (LS-F5886, LSBio), Ang1-7 (CSB-E14242h).

Techniques: Protein-Protein interactions, Activation Assay, Transmission Assay

Journal: Circulation Research

Article Title: Mineralocorticoid Receptor Blocker Increases Angiotensin-Converting Enzyme 2 Activity in Congestive Heart Failure Patients

doi: 10.1161/01.res.0000187500.24964.7a

Figure Lengend Snippet: Figure 1. ACE2 activity assay. Fluo- rescence kinetic of cultured of mac- rophages was measured in the absence (light bars) or presence (dark bars) of AngI. A, Balb/C MPMs cul- tured in 96 wells were washed, and activity of ACE2 was measured in dif- ferent reaction mixtures: Complete reaction mixture (RM), reaction mix- ture without leucine dehydrogenase (RM-LeuDH), and reaction mixture without NAD (RM-NAD). Ang II assay performed in the presence of 7.7 nmol Ang II. B, Fluorescence kinetic was measured in cultured HMDMs (control) and in HMDMs that were preincubated with a specific anti- human ACE2 ectopic domain before activity measurement (anti ACE2 Ab). Results are expressed as meanSEM of quadruplicates.

Article Snippet: Preincubation of human macrophages with a specific antibody directed against the ectopic domain of human ACE2 (R&D Systems) completely abolished the AngI-induced increase in fluorescence (Figure 1B), indicating the association of the AngI degradation activity with ACE2.

Techniques: Activity Assay, Cell Culture, Ii Assay, Fluorescence, Control

Journal: Circulation Research

Article Title: Mineralocorticoid Receptor Blocker Increases Angiotensin-Converting Enzyme 2 Activity in Congestive Heart Failure Patients

doi: 10.1161/01.res.0000187500.24964.7a

Figure Lengend Snippet: Figure 3. Effect of spironolactone ther- apy on ACE and ACE2 in HMDMs. HMDMs were isolated from blood sam- ples obtained from CHF patients before and after spironolactone therapy. Enzy- matic activities were measured in intact cells in culture and normalized to milli- gram cell protein. A, ACE2 activity (AngI- induced ACE2-mediated release of leucine) expressed in pmol per minute. B, ACE activity expressed in units (n6). Densitometric analysis of reverse- transcription PCR results for ACE2 (n10) and ACE (n9) mRNA expres- sion, normalized to -actin, are present- ed in C and D, respectively. C, insert, Representative gel of ACE 2 and -actin of 3 CHF patients before and after spi- ronolactone therapy. Values are meanSEM. *P0.01, **P0.005 after vs before spironolactone.

Article Snippet: Preincubation of human macrophages with a specific antibody directed against the ectopic domain of human ACE2 (R&D Systems) completely abolished the AngI-induced increase in fluorescence (Figure 1B), indicating the association of the AngI degradation activity with ACE2.

Techniques: Isolation, Activity Assay, Reverse Transcription

Journal: Circulation Research

Article Title: Mineralocorticoid Receptor Blocker Increases Angiotensin-Converting Enzyme 2 Activity in Congestive Heart Failure Patients

doi: 10.1161/01.res.0000187500.24964.7a

Figure Lengend Snippet: Figure 4. Effect of eplerenone treatment on ACE and ACE2 in mice. Balb/C mice were randomly divided into 2 groups: Eplerenone-treated (200 mg kg1 d1 in their drinking water for 2 weeks or control) and untreated. MPMs were harvested and cultured, and hearts and kidneys were homogenized. A, Aliquots of the nonsolubilized homogenate pellet were taken for ACE (light bars) and ACE2 (dark bars) activity measurements. Results are expressed as the ratio of activity measured in eplerenone-treated mice to control untreated mice. ACE2 con- trol activities in MPMs, hearts, and kidneys were 1584, 58, and 3063 fmol leucine formation per minute, respectively. Each bar represents meanSEM. n4. NS indicates non significant, eplerenone vs control. *P0.001.

Article Snippet: Preincubation of human macrophages with a specific antibody directed against the ectopic domain of human ACE2 (R&D Systems) completely abolished the AngI-induced increase in fluorescence (Figure 1B), indicating the association of the AngI degradation activity with ACE2.

Techniques: Control, Cell Culture, Activity Assay

Journal: Circulation Research

Article Title: Mineralocorticoid Receptor Blocker Increases Angiotensin-Converting Enzyme 2 Activity in Congestive Heart Failure Patients

doi: 10.1161/01.res.0000187500.24964.7a

Figure Lengend Snippet: Figure 5. The direct effects of aldosterone and its antagonist eplerenone on control of ACE and ACE2 in cultured macrophages. MPMs obtained from C57Bl or p47ko mice were grown in a serum-free medium for 36 hours with no treatment (left), in the presence of 250 nmol aldosterone (middle) or 250 nmol aldosterone 2 mol/L eplerenone (right). Enzymatic activities were measured in intact cells in culture and normalized to milligram cell protein. A, Activity of ACE2 (AngI-induced ACE2-mediated release of leucine) expressed in pmol per minute, B, ACE activity expressed in units. Densitometric analysis of reverse transcription PCR results for ACE2 and ACE mRNA expression, normalized to -actin, are presented in C and D, respectively. Each bar represents meanSEM (n4). *P0.05 al- dosterone vs control; **P0.05 eplerenone vs aldosterone.

Article Snippet: Preincubation of human macrophages with a specific antibody directed against the ectopic domain of human ACE2 (R&D Systems) completely abolished the AngI-induced increase in fluorescence (Figure 1B), indicating the association of the AngI degradation activity with ACE2.

Techniques: Control, Cell Culture, Activity Assay, Reverse Transcription, Expressing

Journal: Circulation Research

Article Title: Mineralocorticoid Receptor Blocker Increases Angiotensin-Converting Enzyme 2 Activity in Congestive Heart Failure Patients

doi: 10.1161/01.res.0000187500.24964.7a

Figure Lengend Snippet: Figure 6. The effects of NADPH oxidase and ROS inhibition on aldosterone-induced control of ACE and ACE2 in cultured macrophages. MPMs obtained from C57Bl mice were grown in a serum-free medium for 36 hours with no treat- ment (control) or in the presence of 250 nmol/L aldosterone (Aldo), 250 nmol/L aldosterone 30 mg/mL (SODAldo), or 250 nmol/L aldosterone 50 apocynin (ApocyninAldo). Enzymatic activities were measured in intact cells in culture and normalized to milligram cell protein. A, Activ- ity of ACE2 (AngI-induced ACE2-mediated release of leucine) expressed in pmol per minute. B, ACE activity expressed in units. Each bar rep- resents meanSEM (n4). *P0.05 vs control; **P0.05 vs aldosterone).

Article Snippet: Preincubation of human macrophages with a specific antibody directed against the ectopic domain of human ACE2 (R&D Systems) completely abolished the AngI-induced increase in fluorescence (Figure 1B), indicating the association of the AngI degradation activity with ACE2.

Techniques: Inhibition, Control, Cell Culture, Activity Assay

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Construction of HEK293T cells line that continuously expressing ACE2-GFP. HEK293Tcells were transfected with pCMV-ACE2-GFPSark tag plasmid and selected with hygromycin B to generate cell lines expressingACE2-GFP, cells were passaged 10 times to ensure stable expression. a Confocal imaging of HEK293T cell line with stably expressed ACE2-GFP. b Cell lysates were analyzed by Western blot with antibodies specific for ACE2 to confirm the expression of ACE2-GFP in HEK293T cells

Article Snippet: HEK293T/ACE2 stable cell line was generated by transducing HEK293T cells with pCMV-Flag-ACE2,(Sino Biological, HG10108-NF), and 1 × 106 cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate), supplemented with protease inhibitors.

Techniques: Expressing, Transfection, Plasmid Preparation, Imaging, Stable Transfection, Western Blot

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Establishment of the in vitro cell capturing system using immobilized spikeS1 protein. a - e , Optimization of the dose of immobilized spike S1 protein for capturing HEK293T/ACE2-GFP cells. Different amounts of S1 protein were coated on the 96-well microplate to capture the HEK293T/ACE2-GFP cells. Representative micrographs of captured cells are shown for 0 μg (A),0.125 μg(B),0.25 μg (C),0.5 μg(D),and 1.0 μg(E). F Quantification of captured cells using CCK8 test, data are presented as mean ± SD

Article Snippet: HEK293T/ACE2 stable cell line was generated by transducing HEK293T cells with pCMV-Flag-ACE2,(Sino Biological, HG10108-NF), and 1 × 106 cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate), supplemented with protease inhibitors.

Techniques: In Vitro

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Competitive curve of cell capturing inhibition by RBD domain of Spike protein. 0.5 μg Spike S1 protein was coated on the microplate to capture the HEK293T/ACE2-GFP cells in presence of different concentrations of spike RBD protein. Amount of captured cells were determined by CCK8 test and expressed as relative value by setting the capability to capture cells of the non-RBD group as 100%. Data were plotted with a four-parameter logistic (4PL) regression curve fit of relative cell number (y-axis) versus the competitor concentration (x-axis)

Article Snippet: HEK293T/ACE2 stable cell line was generated by transducing HEK293T cells with pCMV-Flag-ACE2,(Sino Biological, HG10108-NF), and 1 × 106 cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate), supplemented with protease inhibitors.

Techniques: Inhibition, Concentration Assay

Journal: Biomolecules & Therapeutics

Article Title: Primary Cilium by Polyinosinic:Polycytidylic Acid Regulates the Regenerative Migration of Beas-2B Bronchial Epithelial Cells

doi: 10.4062/biomolther.2022.009

Figure Lengend Snippet: Beas-2B cell migration was decreased by the treatment with SARS-CoV-2 (2019-nCoV) spike protein. (A, B) Beas-2B cells were transfected with pCMV control or pCMV3-ACE2-Flag plasmids DNA for 24 h. Cells were treated with 25 ng/mL SARS-CoV-2 (2019-nCoV) spike protein in the presence or absence of FBS for another 24 h. The cells were fixed and stained with antibody against Arl13b and DAPI. The ciliated cells were counted (n>500 cells) (A). Cell lysates were prepared and the protein expression of ACE2-Flag was detected by western blotting using antibody against Flag (B). (C-E) Beas-2B cells were plated on 35-mm 2 dishes and incubated for 24 h. A confluent monolayer of Beas-2B cells was then scratched with a sterile pipet tip. Then, cells were treated with 25 ng/mL spike protein and incubated for 24 h. Migration of cells into the space left by the scratch was photographed using a phase-contrast microscope at 200× magnification (C). Percentage of cell migration was quantified by subtracting the empty area remained at each time point from that at the initiation using NIH image analysis software (version 1.62; National Institutes of Health), and compared to that of the 0-h time point. Percentage of cell migration was presented as bar graph (D). Changes in single cell path were analyzed by tracking program, ImageJ plugin MTrackJ (Version 1.5.0). Movement track was presented in coordinates for each cell on a polar grid with the normalization of start points to the origin (E). Data in bar graphs represented as means ± SD. # p <0.05, significantly different from pCMV-transfected and spike protein-untreated group incubated with or without FBS & p <0.05, significantly different from pCMV3-ACE2-transfected and spike protein-untreated group incubated with or without FBS (A, D). ** p <0.01, significantly different from spike protein-untreated group (D).

Article Snippet: Human pCMV3-ACE2-Flag plasmid (HG10108-CF) and SARS-CoV-2 (2019-nCoV) spike protein (40592-V08B-B) were purchased from Sino Biological Inc (Wayne, PA, USA).

Techniques: Migration, Transfection, Staining, Expressing, Western Blot, Incubation, Microscopy, Software

Journal: Nature Communications

Article Title: Molecular insights into receptor binding of recent emerging SARS-CoV-2 variants

doi: 10.1038/s41467-021-26401-w

Figure Lengend Snippet: BHK cells stably expressing GFP and hACE2 were incubated with His-tagged MERS-CoV RBD, SARS-CoV-2 WT RBD, Alpha RBD, Beta RBD, Gamma RBD, Mink-Y453F RBD, Mink-N501T RBD, and Mink-F486L RBD, respectively. APC anti-His antibodies were used to detect the His-tagged protein binding to the cells. Representative results from three experiments are shown. The mean ± SD percentages of RBD-binding cells in the three experiments are shown in the right lower bar chart. Statistical significance was analyzed using one-way ANOVA with a Tukey’s multiple comparison test for multiple groups.

Article Snippet: Briefly, lentiviruses were packaged in HEK293T cells co-transfected with pLV-C-GFPSpark-hACE2 (Sino Biological) and helper plasmid pLP1, pLP2, and pLP/VSV-G (Invitrogen) at a ratio of 20:20:13:5 using Lipofectamine 2000.

Techniques: Stable Transfection, Expressing, Incubation, Protein Binding, Binding Assay

Journal: Nature Communications

Article Title: Molecular insights into receptor binding of recent emerging SARS-CoV-2 variants

doi: 10.1038/s41467-021-26401-w

Figure Lengend Snippet: a – k Mouse Fc (mFc)-fused hACE2 or miACE2 in the supernatant was captured in the CM5 chip via its interaction with the pre-immobilized anti-mFc antibody. Various concentrations of SARS-CoV-2 WT RBD ( a ), Alpha RBD ( b ), Beta RBD ( c ), Gamma RBD ( d ), Mink-Y453F RBD ( e ), Mink-N501T RBD ( f ), and Mink-F486L RBD ( g ) protein were used to evaluate their binding affinity for hACE2. Serially diluted WT RBD ( h ), Mink-Y453F RBD ( i ), Mink-N501T RBD ( j ), and Mink-F486L RBD ( k ) protein were measured the binding to miACE2. K D , ka, and kd values are all recorded and the representative results from three experiments are shown. The data are presented as the mean ± SEM of three independent replicates ( n = 3).

Article Snippet: Briefly, lentiviruses were packaged in HEK293T cells co-transfected with pLV-C-GFPSpark-hACE2 (Sino Biological) and helper plasmid pLP1, pLP2, and pLP/VSV-G (Invitrogen) at a ratio of 20:20:13:5 using Lipofectamine 2000.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Molecular insights into receptor binding of recent emerging SARS-CoV-2 variants

doi: 10.1038/s41467-021-26401-w

Figure Lengend Snippet: a – e The WT RBD-hACE2 structure (PDB: 6LZG) is shown in the center. Superimposition of WT RBD-hACE2 and each variant RBD-hACE2 (including Beta RBD-hACE2 ( a ), Gamma RBD-hACE2 ( b ), Alpha RBD-hACE2 ( c ), Mink-F486L RBD-hACE2 ( d ), Mink-Y453F RBD-hACE2 ( e )) are shown in each surrounding panel. In each structure the hACE2 is colored in light pink. SARS-CoV-2 WT RBD, Beta RBD, Gamma RBD, Alpha RBD, Mink-F486L RBD, and Mink-Y453F RBD are colored in gray, cyan, orange, yellow, green, and magenta, respectively. The key contact residues are shown as stick structures and labeled appropriately. The cation-π interaction, π-π stacking interaction, salt bridge, and hydrogen bonds are colored in magenta, blue, orange, and yellow, respectively. Hydrogen bond interactions were analyzed at a cutoff of 3.5 Å. f The detailed hydrogen bonds between the Mink-Y453F RBD and hACE2 are shown.

Article Snippet: Briefly, lentiviruses were packaged in HEK293T cells co-transfected with pLV-C-GFPSpark-hACE2 (Sino Biological) and helper plasmid pLP1, pLP2, and pLP/VSV-G (Invitrogen) at a ratio of 20:20:13:5 using Lipofectamine 2000.

Techniques: Variant Assay, Labeling