Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Susceptibility of various cell lines to CPE following infection by HCoVs

Article Snippet: The following antibodies and isotypes were used to quantify the surface marker expression and receptor modulation in the uninfected (control) and infected cells: PE anti-human CD13, anti-human CD147 (isotype-control antibody mouse IgG1 κ, BioLegend); and anti-human CD326 (isotype-control antibody mouse IgG2a κ; BioLegend); human anti-ACE2 antibody (isotype-control antibody Goat IgG; R&D Systems) and anti-GD3, 9-O-acetyl (clone UM4D4; isotype-control antibody mouse IgM κ; Millipore Sigma).

Techniques: Infection

Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Phenotypic characterization of cell surface markers of OC43-infected cells. Phenotypic characterization of ( A ) uninfected (blue) and ( B ) OC43-infected HCT-8 cells (red). CD13, CD147, CD326, ACE2, and GD3 expression were probed by flow cytometry. ( C ) Histogram plots of control and infected HCT-8 cells relative to isotype control (gray). Histograms plots showing ( D ) percentage of cells expressing a given marker and ( E ) mean fluorescence intensity. Data in panels D and E represent the mean of two biological replicates for all antigens except GD3, where three biological replicates were carried out. Statistical analysis was performed with the multiple unpaired t -test with Welch’s correction. Only results that reached statistical significance were identified: * P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.0001, **** P ≤ 0.0001.

Article Snippet: The following antibodies and isotypes were used to quantify the surface marker expression and receptor modulation in the uninfected (control) and infected cells: PE anti-human CD13, anti-human CD147 (isotype-control antibody mouse IgG1 κ, BioLegend); and anti-human CD326 (isotype-control antibody mouse IgG2a κ; BioLegend); human anti-ACE2 antibody (isotype-control antibody Goat IgG; R&D Systems) and anti-GD3, 9-O-acetyl (clone UM4D4; isotype-control antibody mouse IgM κ; Millipore Sigma).

Techniques: Infection, Expressing, Flow Cytometry, Control, Marker, Fluorescence

Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Phenotypic characterization of cell surface markers of 229E-infected cells. Phenotypic characterization of ( A ) uninfected (blue) and ( B ) 229E-infected MRC-5 cells (red). CD13, CD147, CD326, and ACE2 expression were probed by flow cytometry. ( C ) Histogram plots of control and infected MRC-5 cells relative to isotype control (gray). Histograms plots showing ( D ) percentage of cells expressing a given marker and ( E ) mean fluorescence intensity. Data in panels D and E represent the mean of three biological replicates, except for CD147 where two biological replicates were carried out. Statistical analysis was performed with the multiple unpaired t -test with Welch’s correction. Only results that reached statistical significance were identified: * P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.0001, **** P ≤ 0.0001.

Article Snippet: The following antibodies and isotypes were used to quantify the surface marker expression and receptor modulation in the uninfected (control) and infected cells: PE anti-human CD13, anti-human CD147 (isotype-control antibody mouse IgG1 κ, BioLegend); and anti-human CD326 (isotype-control antibody mouse IgG2a κ; BioLegend); human anti-ACE2 antibody (isotype-control antibody Goat IgG; R&D Systems) and anti-GD3, 9-O-acetyl (clone UM4D4; isotype-control antibody mouse IgM κ; Millipore Sigma).

Techniques: Infection, Expressing, Flow Cytometry, Control, Marker, Fluorescence

Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Phenotypic characterization of cell surface markers of NL63-infected cells. Phenotypic characterization of ( A ) uninfected (blue) and ( B ) NL63-infected LLC-MK2 cells (red). CD13, CD147, CD326, and ACE2 expression were probed by flow cytometry. ( C ) Histogram plots of control and infected LLC-MK2 cells relative to isotype control (gray). Histograms plots showing ( D ) percentage of cells expressing a given marker and ( E ) mean fluorescence intensity. Data in panels D and E represent the mean of three biological replicates, except for CD13 where two biological replicates were carried out. Statistical analysis was performed with the multiple unpaired t -test with Welch’s correction. Only results that reached statistical significance were identified: * P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.0001, **** P ≤ 0.0001.

Article Snippet: The following antibodies and isotypes were used to quantify the surface marker expression and receptor modulation in the uninfected (control) and infected cells: PE anti-human CD13, anti-human CD147 (isotype-control antibody mouse IgG1 κ, BioLegend); and anti-human CD326 (isotype-control antibody mouse IgG2a κ; BioLegend); human anti-ACE2 antibody (isotype-control antibody Goat IgG; R&D Systems) and anti-GD3, 9-O-acetyl (clone UM4D4; isotype-control antibody mouse IgM κ; Millipore Sigma).

Techniques: Infection, Expressing, Flow Cytometry, Control, Marker, Fluorescence

Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Soluble ACE2 protein and anti-ACE2 monoclonal blocking antibody inhibit NL63 infection in LLC-MK2 cells. ( A ) Treatment with soluble ACE2 or control protein ovalbumin at 5 and 10 mg/mL was pre-incubated with NL63 virus at MOI 0.1 (virus titer: 8.89 × 10 5 TCID 50 /mL) for 1 hour, and the protein and virus mixture complex was added to the LLC-MK2 cells. ( B ) Similarly, cells were incubated for 1 hour with human anti-ACE2 monoclonal blocking antibody (10, 40, and 120 µg/mL) or unrelated control antibody (anti-DC-SIGN; 40 µg/mL) followed by addition of NL63 virus (MOI 0.1). Five days post-infection, both culture supernatants and cell lysates were harvested for quantification of nucleocapsid (N) and spike (S) vRNA copies by ddPCR. Percent relative viral inhibition following treatment with (C) sACE2 or (D) anti-ACE2 monoclonal antibody compared to their respective untreated control. Data represent three biological replicates for sACE2 or four (anti-ACE2) with three technical replicates per experiment. Statistical analysis was performed using an unpaired t -test with Welch’s correction ( P < 0.05) relative to untreated control. n.s. denoted as no statistical difference; * P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.0001, **** P ≤ 0.0001.

Article Snippet: The following antibodies and isotypes were used to quantify the surface marker expression and receptor modulation in the uninfected (control) and infected cells: PE anti-human CD13, anti-human CD147 (isotype-control antibody mouse IgG1 κ, BioLegend); and anti-human CD326 (isotype-control antibody mouse IgG2a κ; BioLegend); human anti-ACE2 antibody (isotype-control antibody Goat IgG; R&D Systems) and anti-GD3, 9-O-acetyl (clone UM4D4; isotype-control antibody mouse IgM κ; Millipore Sigma).

Techniques: Blocking Assay, Infection, Control, Incubation, Virus, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: tRNA Modifications: A Tale of Two Viruses—SARS-CoV-2 and ZIKV

doi: 10.3390/ijms26157479

Figure Lengend Snippet: Elongator complex loss reduces SARS-CoV-2 and ZIKV infection. ( A ) Schematic representation of U34 tRNA modification pathway in FD (ELP1-deficient) cells. The Western blot shows ACE2 receptor and actin levels in cells transduced by ACE2-expressing lentivector (VLP ACE2 ). ( B ) Quantification of U34 modification levels (ncm 5 U, mcm 5 U, and mcm 5 s 2 U) in wild-type ( wt ) versus FD cells determined by mass spectrometry on tRNA-enriched RNA fractionations. ( C ) SARS-CoV-2 infection levels measured by RT-qPCR in wt and FD cells at different multiplicities of infection (MOI) 24 h post-infection. ( D ) ZIKV infection levels measured by RT-qPCR in wt and FD cells at different MOIs 48 h post-infection.

Article Snippet: Seventy-two hours after transduction, accurate ACE2 expression was controlled by Western blots using anti-ACE2 antibody (Human ACE-2 Antibody, AF933, R&D systems (a Bio-Techne brand (Minneapolis, MN, USA)).

Techniques: Infection, Modification, Western Blot, Expressing, Mass Spectrometry, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: tRNA Modifications: A Tale of Two Viruses—SARS-CoV-2 and ZIKV

doi: 10.3390/ijms26157479

Figure Lengend Snippet: ALKBH8 and CTU1 knockdown individually impairs SARS-CoV-2 and ZIKV infection. ( A ) Schematic showing shRNA-mediated knockdown approach targeting U34 ALKBH8 and CTU1 components of U34 modification pathway. The Western blot analysis of ACE2 expression in A549 cells with and without lentiviral vector expressing ACE2 (VLP ACE2 ). ( B ) Western blot validation of ALKBH8 and CTU1 knockdown efficiency with corresponding actin loading controls. Numbers indicate relative protein levels. ( C ) SARS-CoV-2 infection levels measured by RT-qPCR following knockdown of the indicated genes 24 h post-infection. ( D ) ZIKV infection levels measured by RT-qPCR following knockdown of the indicated genes 48 h post-infection.

Article Snippet: Seventy-two hours after transduction, accurate ACE2 expression was controlled by Western blots using anti-ACE2 antibody (Human ACE-2 Antibody, AF933, R&D systems (a Bio-Techne brand (Minneapolis, MN, USA)).

Techniques: Knockdown, Infection, shRNA, Modification, Western Blot, Expressing, Plasmid Preparation, Biomarker Discovery, Quantitative RT-PCR

Journal: Biochemical and Biophysical Research Communications

Article Title: Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor

doi: 10.1016/j.bbrc.2004.05.114

Figure Lengend Snippet: (A) Expression of ACE2 in cell culture cell lines susceptible (+) or refractory (−) to SARS-CoV infection. Total RNA was isolated from the indicated cell lines followed by reverse transcription. Subsequently, a nested PCR with ACE2-specific oligonucleotides was performed using either the resulting cDNAs as templates (middle panel, +RT) or employing the input RNA (upper panel, −RT). As a control, all cDNAs were subjected to a PCR with GAPDH-specific oligonucleotides (lower panel). (B) Enhanced SARS-CoV S-mediated entry into 293T cells transiently over-expressing ACE2. ACE2 of human (hu) and African green monkey (agm) origin or human CD13 were transiently expressed in 293T cells followed by infection with SARS-CoV S-pseudotypes carrying a luciferase reporter gene. After 72 h, cells were lysed and luciferase activity was determined in the cell extracts. Each experiment was performed in quadruplicate and repeated at least three times with independent virus stocks.

Article Snippet: ACE2-proteins were detected using a monoclonal ACE2 antibody (R&D systems, Minneapolis).

Techniques: Expressing, Cell Culture, Infection, Isolation, Reverse Transcription, Nested PCR, Control, Luciferase, Activity Assay, Virus

Journal: Biochemical and Biophysical Research Communications

Article Title: Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor

doi: 10.1016/j.bbrc.2004.05.114

Figure Lengend Snippet: Analysis of the contribution of the ACE2 cytoplasmic domain to receptor function. (A) Schematic overview depicting the C-terminal ACE2 mutants analyzed. Putative tyrosine and casein kinase motifs are boxed. (B) Surface expression of ACE2 and the ACE2 deletion mutants. ACE2 was transiently expressed in 293T cells and analyzed by FACS using a polyclonal ACE2 antiserum followed by incubation with a polyclonal FITC-labeled secondary antibody (left panel, dark grey). As controls, pcDNA3-transfected cells were incubated with the secondary antibody (black line) or with both the ACE2-specific antiserum in combination with the secondary antibody (light grey). Similarly, the indicated ACE2 deletion mutants were subjected to FACS analysis; the percentage of ACE2 expressing cells is shown (middle panel). In parallel, expression of wild type ACE2 (lane 2) and all ACE2 mutants was examined by Western blot analysis (right panel: lane 1, pcDNA3; lane 3, mutant 1–790; lane 4, mutant 1–779; lane 5, mutant 1–775; and lane 6, mutant 1–771). (C) Role of the cytoplasmic domain within ACE2 for SARS-CoV S-mediated infection of target cells. ACE2 and the indicated deletion mutants were transiently expressed in 293T cells followed by infection with S-pseudotypes carrying luciferase as reporter gene. After 72 h, the luciferase activity was determined. Each experiment was performed in quadruplicate and repeated at least three times with independent virus preparations.

Article Snippet: ACE2-proteins were detected using a monoclonal ACE2 antibody (R&D systems, Minneapolis).

Techniques: Expressing, Incubation, Labeling, Transfection, Western Blot, Mutagenesis, Infection, Luciferase, Activity Assay, Virus

Journal: Biochemical and Biophysical Research Communications

Article Title: Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor

doi: 10.1016/j.bbrc.2004.05.114

Figure Lengend Snippet: Expression of soluble ACE2 protein and inhibition of SARS-CoV S-driven infection. (A) Expression of the soluble ACE2 ectodomain. Either a pcDNA3 control vector (lane 1), wild type ACE2 (lane 2) or an ACE2 variant comprising only the ectodomain (lane 3) was transiently expressed in 293T cells. After 48 h, cells and culture supernatants (lanes 4–6) were harvested and analyzed for ACE2 expression via Western blot. (B) Inhibition of S-mediated entry into 293T cells by soluble ACE2. S-bearing pseudotypes and VSV-G pseudotypes normalized for equal luciferase activity (10 4 c.p.s.) upon infection of target cells were pre-incubated with the indicated dilutions of concentrated soluble ACE2 and used for infection of 293T cells. Luciferase activity was determined in cell extracts after 72 h. The relative luciferase units obtained after infection in the absence of soluble ACE2 was set as 100%. Each experiment was performed in quadruplicate and repeated three times; similar results were obtained with a different soluble ACE2 preparation and with independent virus stocks.

Article Snippet: ACE2-proteins were detected using a monoclonal ACE2 antibody (R&D systems, Minneapolis).

Techniques: Expressing, Inhibition, Infection, Control, Plasmid Preparation, Variant Assay, Western Blot, Luciferase, Activity Assay, Incubation, Virus

Journal: International Journal of Molecular Sciences

Article Title: Distinctive Properties of Endothelial Cells from Tumor and Normal Tissue in Human Breast Cancer

doi: 10.3390/ijms22168862

Figure Lengend Snippet: Characterization of ECs by flow cytometry markers. The cells were stained for the following markers: VWF, ACE, CD31, CD34, CD133, CD105, PDPN, AP, αSMA, EGFR. Data were recorded for 10,000 events using CellQuest software (v.2.3.0.84) and presented as histogram overlays. ( A ) The most characteristic markers for ECs and EPCs. ( B ) Markers associated with cancerous phenotype. Histogram overlays display representative repetitions; gray—healthy ECs unstained, black—tumor ECs unstained, green—healthy ECs stained, red—tumor ECs stained. Y axis = the number of events; X axis = fluorescence intensity; the bar charts present delta MFI. Ns—not significant; * p < 0.05 in Student t test vs. HBH.MEC. Data are reported as the means ± SEM (n = 3).

Article Snippet: The following antihuman antibodies were used: APC-conjugated EGFR Antibody (BioLegend, San Diego, CA, USA #352905), PerCP-conjugated Podoplanin Antibody (eBioscience, San Diego, CA, USA #46-9381-42), PE-Cy7-conjugated CD34 Antibody (BD Biosciences, Franklin Lakes, NJ, USA #560710), PE-conjugated CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany#130-098-046), FITZ-conjugated CD105 (BioLegend, San Diego, CA, USA #323204), PE-conjugated Anti-CD309 Antibody (Beckman Coulter Life Sciences, Marseille Cedex, France #a64615), Alexa Fluor 700-conjugated BCL-6 Antibody (BD Biosciences, Franklin Lakes, NJ, USA # 566993), PE-conjugated Anti-CD62L Antibody (Beckman Coulter Life Sciences, Marseille Cedex, France #IM2214U), FITC-conjugated Anti-CD54 Antibody (Beckman Coulter Life Sciences, Marseille Cedex, France #IM0726U), KO525-A-conjugated Anti-CD31 Antibody (BD Horizon, Franklin Lakes, NJ, USA #563454), Alexa Fluor 488-conjugated Alkaline Phosphatase (BD Biosciences, Franklin Lakes, NJ, USA #561495), VWF Antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA #sc-53466), ACE Antibody (R&D Systems Minneapolis, MA, USA #AF929).

Techniques: Flow Cytometry, Staining, Software, Fluorescence