Journal: PLoS ONE

Article Title: Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii

doi: 10.1371/journal.pone.0257089

Figure Lengend Snippet: (A) HEK-cell produced RBD fragment fused to Rabbit IgG Fc fragment (RBD::rFc) binding to immobilized biotinylated recombinant human ACE2 was detected by anti-Rabbit IgG-HRP antibodies and TMB chromogenic reaction. Data shown represent values from one experiment. (B) ACE2 Receptor binding competition assay between a constant concentration of partially purified ER-Golgi Retained Algae-Produced RBD::mClover (~40 nM) and increasing amounts of RBD::rFc or Bovine Serum Albumin showing specific competition. RBD::mClover binding was detected using anti-GFP HRP antibodies. Data points represent mean and error bars represent Standard Error of the Mean of normalized A 490 signal values over three independent experimental repeats.

Article Snippet: Strepavidin coated microtiter plates (Cat#15124, Thermo Fisher) were washed 3X in Receptor Assay Blocking buffer (25 mM TrisHCl, 150mM NaCl, pH 7.4, 0.1% wt/vol Bovine Serum Albumin, 0.05% vol/vol Tween-20) and then were coated with 50 ng per well of biotylated human ACE2 produced in HEK cells lines (Cat#10108-H08H-B Sino Biological) dissolved in 100 μL of PBS for one hour at room temperature with gentle shaking on an orbital table.

Techniques: Produced, Binding Assay, Recombinant, Competitive Binding Assay, Concentration Assay, Purification

Journal: Journal of Virology

Article Title: Variations in Cell Surface ACE2 Levels Alter Direct Binding of SARS-CoV-2 Spike Protein and Viral Infectivity: Implications for Measuring Spike Protein Interactions with Animal ACE2 Orthologs

doi: 10.1128/jvi.00256-22

Figure Lengend Snippet: Recombinant SARS-CoV-2 RBD protein binds to human ACE2 and facilitates pseudoviral infectivity. (A) Schematic of the bicistronic mRNA (3,537 bp) for the production of human ACE2 (2,415 bp; 805 amino acids [aa]) and the reporter cell surface protein mouse Thy1.1 (489 bp; 163 aa). (B) HRT-18G cells were stably transfected with a plasmid containing cDNA of hACE2 and magnetically sorted for Thy1.1 expression. HRT-18G/hACE2 (blue trace) and parental HRT-18G (shaded histogram) cells were stained with fluorescent anti-Thy1.1 antibody, and expression was analyzed by flow cytometry. (C) HRT-18G cells stably expressing ACE2 (blue trace) and parent cells (shaded histogram) were incubated with fluorescent anti-ACE2 antibody and analyzed by flow cytometry. (D) HRT-18G parental cells (shaded histogram) and HRT-18G/hACE2 cells (blue trace) were incubated with the Alexa Fluor 647-labeled SARS-CoV-2 S-protein RBD and analyzed for protein binding by flow cytometry. (E) Same as panel D except that cells were incubated with a range of concentrations of the Alexa Fluor 647-labeled SARS-CoV-2 S-protein RBD and analyzed by flow cytometry. (F) HRT-18G/hACE2 and HRT-18G cells were infected with GFP-expressing SARS-CoV-2 pseudovirus at an MOI of 10 for the indicated times, washed to remove excess virus, and incubated at 37°C overnight. Infectivity was analyzed by flow cytometry 16 h later, and the percentage of GFP-positive (GFP + ) cells is reported. (G) HRT-18G/hACE2 and parent cells were infected with a GFP-expressing SARS-CoV-2 pseudovirus at an MOI of 0.5 and cultured for 36 h prior to analysis by fluorescence microscopy. Cells were stained with DAPI to delineate the nucleus. All data presented are representative of results from three independent experiments. Statistical significance is indicated (***, P < 0.0001). MFI, mean fluorescence intensity.

Article Snippet: The following primary antibodies were used for Western blot analysis: mouse monoclonal β-actin (clone AC-15; Sigma), rabbit polyclonal ACE2 (catalog number 21115-1-AP; Thermo Fisher), mouse monoclonal E-cadherin (clone 4A2C7; Life Technologies), and rabbit polyclonal LC3B (clone D11; Cell Signaling).

Techniques: Recombinant, Infection, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Staining, Flow Cytometry, Incubation, Labeling, Protein Binding, Virus, Cell Culture, Fluorescence, Microscopy

Journal: Journal of Virology

Article Title: Variations in Cell Surface ACE2 Levels Alter Direct Binding of SARS-CoV-2 Spike Protein and Viral Infectivity: Implications for Measuring Spike Protein Interactions with Animal ACE2 Orthologs

doi: 10.1128/jvi.00256-22

Figure Lengend Snippet: Increased ACE2 expression potentiates SARS-CoV-2 RBD binding and pseudoviral infectivity. (A) HRT-18G cells were transiently transfected with cDNA encoding hACE2 and Thy1.1 in a bicistronic cassette, and 2 days later, they were stained with fluorescently labeled anti-Thy1.1 and -ACE2 antibodies and analyzed by flow cytometry. Single-antibody labeling controls are also depicted. (B) Cells were stained with fluorescently labeled anti-Thy1.1 antibody and the Alexa Fluor 647-labeled SARS-CoV-2 S-protein RBD and analyzed by flow cytometry. Histograms of individual protein-labeled cells are also depicted. (C to E) HRT-18G/hACE2 cells (blue traces) were fluorescently sorted based on Thy1.1 expression to generate the cell line HRT-18G/hACE2++ (orange traces). HRT-18G/hACE2 (H) and HRT-18G/hACE2++ (H ++ ) cells and parental HRT-18G cells (P) (shaded histograms) were then incubated with anti-Thy1.1 (C) or anti-ACE2 (D) antibody or the Alexa Fluor 647-labeled SARS-CoV-2 S-protein RBD (E) and analyzed by flow cytometry. Histograms and quantitative MFI measurements are representative of results from three independent biological replicates. (F) HRT-18G/hACE2 and HRT-18G/hACE2++ cells were infected with GFP-expressing SARS-CoV-2 pseudovirus for the indicated times, washed to remove excess virus, incubated in complete medium for 16 h, and analyzed by flow cytometry for GFP expression. All data presented are representative of results from three independent biological experiments. Statistical significance is indicated (***, P < 0.0001).

Article Snippet: The following primary antibodies were used for Western blot analysis: mouse monoclonal β-actin (clone AC-15; Sigma), rabbit polyclonal ACE2 (catalog number 21115-1-AP; Thermo Fisher), mouse monoclonal E-cadherin (clone 4A2C7; Life Technologies), and rabbit polyclonal LC3B (clone D11; Cell Signaling).

Techniques: Expressing, Binding Assay, Infection, Transfection, Staining, Labeling, Flow Cytometry, Antibody Labeling, Incubation, Virus

Journal: Journal of Virology

Article Title: Variations in Cell Surface ACE2 Levels Alter Direct Binding of SARS-CoV-2 Spike Protein and Viral Infectivity: Implications for Measuring Spike Protein Interactions with Animal ACE2 Orthologs

doi: 10.1128/jvi.00256-22

Figure Lengend Snippet: Human (h), feline (f), and mouse (m) ACE2 orthologs are glycosylated, metabolically stable, and detected at the cell surface in stably transfected HRT-18G cells. (A) HRT-18G cells were stably transfected with IRES-Thy1.1 plasmids containing cDNAs of human (blue), feline (orange), and mouse (purple) ACE2 and magnetically sorted until equivalent levels of the reporter protein Thy1.1 were expressed on all three cell lines. Staining of parental HRT-18G cells is shown as a negative control (shaded histogram). (B) RNA was isolated from equal numbers of HRT-18G/hACE2, HRT-18G/fACE2, and HRT-18G/mACE2 cells. cDNAs were made from extracted RNAs of the tested cell lines, and qPCR was performed using GAPDH as a housekeeping gene. C T values from ACE2 amplifications were normalized to C T values from GAPDH, and the average fold changes from three independent experiments are shown. (C) To confirm the specificity of the ACE2 antibody for human ACE2, HRT-18G/hACE2 (blue), HRT-18G/fACE2 (orange), HRT-18G/mACE2 (purple), and HRT-18G (shaded histogram) cells were simultaneously incubated with fluorescently labeled ACE2 antibody and analyzed by flow cytometry. (D) HRT-18G cells expressing the indicated ACE2 orthologs were lysed and either mock treated or deglycosylated by incubation with PNGase F. Cell lysates were then resolved by SDS-PAGE and immunoblotted (IB) with polyclonal antibodies to ACE2 or β-actin. Molecular weight markers are indicated. (E) Cells were cultured with CHX for 0, 5, or 10 h, followed by lysis, SDS-PAGE, and Western blot analysis for either ACE2, β-actin, or LC3B. C, control: HRT-18G cells which do not express any ACE2 ortholog. (F) Cell surface proteins were biotinylated prior to cell lysis. Biotin-conjugated proteins were isolated using a streptavidin agarose slurry and analyzed by Western blotting for ACE2, E-cadherin, and β-actin.

Article Snippet: The following primary antibodies were used for Western blot analysis: mouse monoclonal β-actin (clone AC-15; Sigma), rabbit polyclonal ACE2 (catalog number 21115-1-AP; Thermo Fisher), mouse monoclonal E-cadherin (clone 4A2C7; Life Technologies), and rabbit polyclonal LC3B (clone D11; Cell Signaling).

Techniques: Metabolic Labelling, Stable Transfection, Transfection, Staining, Negative Control, Isolation, Incubation, Labeling, Flow Cytometry, Expressing, SDS Page, Molecular Weight, Cell Culture, Lysis, Western Blot, Control

Journal: Journal of Virology

Article Title: Variations in Cell Surface ACE2 Levels Alter Direct Binding of SARS-CoV-2 Spike Protein and Viral Infectivity: Implications for Measuring Spike Protein Interactions with Animal ACE2 Orthologs

doi: 10.1128/jvi.00256-22

Figure Lengend Snippet: SARS-CoV-2 RBD binding and viral infectivity with different ACE2 orthologs. (A) HRT-18G (shaded histogram), HRT-18G/hACE2 (blue trace), HRT-18G/fACE2 (orange trace), and HRT-18G/mACE2 (purple trace) cell lines were incubated with 7.8 ng of the Alexa Fluor 647-labeled SARS-CoV-2 S-protein RBD and analyzed for SARS-CoV-2 S-protein RBD/ACE2 binding by flow cytometry. (B) Same as panel A except that cells were incubated with different concentrations of the Alexa Fluor 647-labeled RBD. The MFI of the population is reported on the y axis. (C) HRT-18G/hACE2, HRT-18G/fACE2, HRT-18G/mACE2, and HRT-18G cells were infected with GFP-expressing SARS-CoV-2 pseudovirus for the specified times, washed to remove excess virus, and incubated for 16 h, and GFP-expressing cells were quantified by flow cytometry. All data presented are representative of results from three independent biological experiments.

Article Snippet: The following primary antibodies were used for Western blot analysis: mouse monoclonal β-actin (clone AC-15; Sigma), rabbit polyclonal ACE2 (catalog number 21115-1-AP; Thermo Fisher), mouse monoclonal E-cadherin (clone 4A2C7; Life Technologies), and rabbit polyclonal LC3B (clone D11; Cell Signaling).

Techniques: Binding Assay, Infection, Incubation, Labeling, Flow Cytometry, Expressing, Virus

Journal: Diagnostics

Article Title: Postmortem Cardiopulmonary Pathology in Patients with COVID-19 Infection: Single-Center Report of 12 Autopsies from Lausanne, Switzerland

doi: 10.3390/diagnostics11081357

Figure Lengend Snippet: Antibodies used for immunofluorescence staining.

Article Snippet: ACE2 , CL4035 , Membranous , Mouse , Atlas Antibodies/ AMAB91262 , 1:1000.

Techniques: Immunofluorescence, Staining

Journal: Diagnostics

Article Title: Postmortem Cardiopulmonary Pathology in Patients with COVID-19 Infection: Single-Center Report of 12 Autopsies from Lausanne, Switzerland

doi: 10.3390/diagnostics11081357

Figure Lengend Snippet: Composition of antibodies used in the multiplexed immunofluorescence panels.

Article Snippet: ACE2 , CL4035 , Membranous , Mouse , Atlas Antibodies/ AMAB91262 , 1:1000.

Techniques: Immunofluorescence

Journal: Diagnostics

Article Title: Postmortem Cardiopulmonary Pathology in Patients with COVID-19 Infection: Single-Center Report of 12 Autopsies from Lausanne, Switzerland

doi: 10.3390/diagnostics11081357

Figure Lengend Snippet: ( A , B ) Multiplex imaging analysis depicting expression of ACE2, the major receptor for SARS-CoV-2, in different cellular compartments of lung tissues in a representative patient with COVID-19 (case #6). ( A ) Red arrows point to ACE2-expressing pneumocytes, defined by double-expression of ACE2 (yellow) and PANCK (white). ( B ) Cyan arrows highlight ACE2-expressing macrophages, defined by double-expression of ACE2 (yellow) and CD68 (red). All images were acquired on the Vectra Polaris microscope using a 20x objective magnification, and zoomed in areas are depicted for visualization of macrophages. ( C ) The was no difference in the relative frequencies (% of total imaged cells) of CD3+ T cells in lung control tissues (non-COVID-19 DAD, n = 5) and COVID-19 lung tissues ( n = 5), expressed as mean values with SD at the scatter dot plots (Mann–Whitney U test, p = 0.87). ( D ) There was no statistically significant difference of the CD4+ to CD8+ T lymphocyte ratio between the two cohorts (Mann–Whitney U test, p = 0.55).

Article Snippet: ACE2 , CL4035 , Membranous , Mouse , Atlas Antibodies/ AMAB91262 , 1:1000.

Techniques: Multiplex Assay, Imaging, Expressing, Microscopy, MANN-WHITNEY

Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: a SARS-CoV-2 replication kinetics in HAE from different donors, HCoV-NL63 was used as a control ( n = 3). b Transepithelial electrical resistance (TEER in Ω cm 2 ) between the apical and basal poles was measured at each time point ( n = 3). c SARS-CoV-2 infected both ciliated cells (72 h pi) and secretory cells (72 h pi). arrows: virus particles, arrowhead: cilium, asterisk: secretory vesicle, insets dashed-line squares indicate magnification of arrowed areas. d Costaining of SARS-CoV-2 N protein (green) with ciliated cell marker β-tubulin-IV (red), goblet cell marker Muc5AC (red), club cell marker CCSP (red), and ACE2 (red) positive cells. HCoV-NL63 N protein (green) staining was used as a control (72 h pi). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Data a , b are the means ± s.d. of three independent biological replicates. Source data a – d are provided as a Source Data file.

Article Snippet: ACE2 , Sino biologicals (10108-T56), rabbit polyclona (1:100).

Techniques: Infection, Marker, Staining

Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: Source of antibodies and dyes with work concentration for immunofluorescence.

Article Snippet: ACE2 , Sino biologicals (10108-T56), rabbit polyclona (1:100).

Techniques: Concentration Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Variable Induction of Pro-inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo

doi: 10.1101/2021.05.26.445843

Figure Lengend Snippet: Cytokine responses in human PBMC induced by various commercial coronavirus spike proteins. ( A ) Rested PBMC were cultured with or without 2.0 μg/mL of raxibacumab (human anti-anthrax PA IgG used as a negative control), MERS S1-Fc, SARS CoV-1 S1-Fc, SARS CoV-2 S1-Fc from Vendor #2 (V#2 S1-Fc) and Vendor #1 (lot 24529-2003, V#1 S1-Fc 2003), and RBD-Fc from Vendor #2 (V#2 RBD-Fc) and Vendor #1 (lot 24530-2003, V#1 RBD-Fc 2003) for 24 hours. ( B ) Rested PBMC were cultured with or without plate-bound streptavidin (STAV) together with or without S1-biotin and RBD-biotin purchased from Vendor #2. The levels of IL-6 and TNFα were measured using the CBA human inflammatory cytokine kit and flow cytometric analysis. IL-8 levels were measured using an ELISA kit. The concentrations of the cytokines were calculated based on the standard curves, and the induction of cytokines were presented as stimulation indices. Data shown are statistical results (mean ± SE) generated from 8 ( A ) or 3 ( B ) healthy donors. Statistical analyses were performed by Excel using a two-tailed, Student’s T-test. *, **, *** and **** depict p < 0.05, 0.01, 0.005 and 0.001, respectively.

Article Snippet: S1-Fc (Catalog# 40591-V02H, lot LC14AP1605), RBD-Fc (Catalog# 40592-V02H, lot LC14MC2602), S1-biotin (Catalog# 40591-V27HB, lot LC14AU101), RBD-biotin (Catalog# 40592-V27B-B, lot MF14AP2302), ACE2 (Catalog# 10108-H08H, lot MB14JN0906), anti-S1 neutralizing antibody (Catalog# 40591-MM43, lot HB14AP2001) and anti-S1 non-neutralizing antibody (Catalog # 40591-MM42, lot HB14AP0701) were also purchased from Sino Biological (Vendor #2).

Techniques: Cell Culture, Negative Control, Enzyme-linked Immunosorbent Assay, Generated, Two Tailed Test

Journal: bioRxiv

Article Title: Variable Induction of Pro-inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo

doi: 10.1101/2021.05.26.445843

Figure Lengend Snippet: LPS co-purifying with spike protein reagents induces proinflammatory cytokine production. ( A ) MERS S1-Fc, SARS CoV-1 S1-Fc, S1-Fc from Vendor #2 (V#2 S1-Fc), S1-Fc from Vendor #1 (lot 24056-2002-2 (V#1 S1-Fc 2002) and lot 24529-2003 (V#1 S1-Fc 2003)), RBD-Fc from Vendor #2 (V#2 RBD-Fc) and RBD-Fc from Vendor #1 (lot 25130-2004, V#1 RBD-Fc 2004), and ( B ) V#2 S1-Fc, V#1 S1-Fc 2002 and streptavidin (STAV) before and after treatment of endotoxin removal were assessed for the levels of endotoxin using the LAL Chromogenic Endotoxin Quantitation Kit. The concentrations of endotoxin are presented as EU/mg protein. Data shown are mean ± SE of the results from three independent experiments. ( C ) Time course of S1-Fc-induced cytokine responses. Rested PBMC were cultured with or without 2.0 μg/mL of S1-Fc (V#2 S1-Fc and V#1 S1-Fc 2002) for 1, 3, 6 or 24 hours. The levels of IL-6, IL-8 and TNFα in the supernatants of cultured PBMC were assessed using the CBA human inflammatory cytokine kit and flow cytometric analysis. ( D, E ) Blockade of S1-Fc-induced cytokine response by an LPS inhibitor, polymyxin B. Rested PBMC were cultured with or without 2.0 μg/mL of S1-Fc (V#2 S1-Fc) in the presence or absence of polymyxin B for 3 hours. The levels of IL-6, IL-8 and TNFα in the supernatants of cultured PBMC were measured using the CBA human inflammatory cytokine kit and flow cytometric analysis. Data shown in ( D ) are a representative of the flow cytometric results from three healthy donors and data shown in ( E ) are mean ± SE of the stimulation index derived from three healthy donors, which is calculated using mean fluorescent intensities (MFI) of the cytokines and the formula: cytokine MFI of treated PBMC/cytokine MFI of untreated PBMC. Statistical analyses were performed using a two-tailed, Student’s T-test. * and ** depict p < 0.05 and 0.01, respectively.

Article Snippet: S1-Fc (Catalog# 40591-V02H, lot LC14AP1605), RBD-Fc (Catalog# 40592-V02H, lot LC14MC2602), S1-biotin (Catalog# 40591-V27HB, lot LC14AU101), RBD-biotin (Catalog# 40592-V27B-B, lot MF14AP2302), ACE2 (Catalog# 10108-H08H, lot MB14JN0906), anti-S1 neutralizing antibody (Catalog# 40591-MM43, lot HB14AP2001) and anti-S1 non-neutralizing antibody (Catalog # 40591-MM42, lot HB14AP0701) were also purchased from Sino Biological (Vendor #2).

Techniques: Quantitation Assay, Cell Culture, Derivative Assay, Two Tailed Test

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet: SARS-CoV-2 infection in male and female k18-hace2 mice over 7 days post-infection (dpi) (A) Body weight loss in response to intranasal inoculation of SARS-CoV-2 (4 × 10 5 TCID50/50μL/mouse). ∗p < 0.05 vs. male. Data represented in median ± IQR. (B) Lung injury scoring assessed on a scale of 0–4 for each of the following criteria: 1) neutrophil numbers in the alveolar space, 2) alveolar septal thickening, 3) number of hyaline membranes, 4) alveolar hemorrhage, and 5) cellular hyperplasia. ∗p < 0.05 vs. Ctrl and male. Data represented in mean ± SEM. (C) Viral RNA levels in oropharyngeal swabs at 2, 4, and 6 dpi. ∗p < 0.05 vs. male. Data represented in median ± IQR. (D–G) Copy numbers of E-gene and RdRp-gene in multiple organ tissues. ∗p < 0.05 vs. Ctrl. Data represented in mean ± SEM. (H) Detection of SARS-CoV-2 nucleocapsid protein (red) in lung tissues. Scale bars, 50 μm (main images) and 20 μm (magnified). (I) Protein expression in lung tissue detected by western blots in vehicle control (C) and at 7 dpi. (J) Concentration of soluble RAGE protein in plasma in vehicle control and at 7 dpi. ∗p < 0.05 vs. other groups. Data represented in mean ± SEM. (K) Correlation of protein levels of ACE2 and ERα in lung tissue of male mice before and after infection at 7 dpi. (L) Estrogen receptor alpha (ERα)/androgen receptor (AR) ratio assessed from western blot analysis in relation to β-actin loading control. ∗p < 0.05 vs. male. Data represented in mean ± SEM. (M) Concentration of 17β Estradiol in lung tissue homogenates in vehicle control and at 7 dpi. ∗p < 0.05 vs. male. n = 5–7 biologically independent mice for all groups. Data represented in mean ± SEM. (see also Figure S1 ).

Article Snippet: The hACE2 concentration in both rabbit and mouse plasma and lung tissue of mice was measured by hACE2 ELISA (#ab235649: Abcam, UK) following the manufacturer’s instructions.

Techniques: Infection, Expressing, Western Blot, Concentration Assay

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet: Inhalation of rACE2 attenuates SARS-CoV-2 infection, replication, and lung injury and recovers expression of endogenous ACE2 and ERα in K18-hACE2 male mice at 7 dpi (A) Study scheme. Mice were intranasally inoculated with SARS-CoV-2 (4 × 10 5 TCID50/50μL/mouse) and 48 h later received daily nebulization of rACE2 (12 mg/kg) or vehicle control solution for 5 days. (B and C) Copy numbers of viral envelope (E) gene by RT-qPCR and titers (TCID50) in lung tissue in the SARS-CoV-2 infection alone and rACE2-treated groups. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (D) Gene expression of IL-6 in lung tissue in the infection alone and rACE2-treated groups. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (E) Concentration of soluble RAGE protein in plasma in infection alone and rACE2-treated mice. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (F) Detection of SARS-CoV-2 nucleocapsid protein (red) in lung tissues. Scale bars, 50 μm (main images) and 20 μm (magnified). (G) Representative lung histology by H&E staining. Arrows indicate neutrophil infiltration in the alveolar space. Lung injury scores from 6 animals per group are also shown. ∗p < 0.05 vs. naive control (C), ♰p < 0.05 vs. SARS-CoV-2 alone & (C). Data represented in mean ± SEM. (H and I) Protein expression in lung tissue detected by western blots in naive control (C), SARS-CoV-2 (CoV2) alone, and rACE2-treated groups. ∗p < 0.05 vs. C & rACE2-treated groups. Data represented in mean ± SEM. (J and K). Fold changes in the concentrations of 17β Estradiol and ERα/AR ratio assessed from western blot analysis in relation to β-actin loading control in lung tissue in naive control (C), SARS-CoV-2 (CoV2) alone, and rACE2-treated groups. ∗p < 0.05 vs. C and SARS-CoV-2, respectively. n = 5–7 biologically independent mice for all groups. Data represented in mean ± SEM. (see also Figure S3 ).

Article Snippet: The hACE2 concentration in both rabbit and mouse plasma and lung tissue of mice was measured by hACE2 ELISA (#ab235649: Abcam, UK) following the manufacturer’s instructions.

Techniques: Infection, Expressing, Quantitative RT-PCR, Concentration Assay, Staining, Western Blot

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet:

Article Snippet: The hACE2 concentration in both rabbit and mouse plasma and lung tissue of mice was measured by hACE2 ELISA (#ab235649: Abcam, UK) following the manufacturer’s instructions.

Techniques: Isolation, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software