Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: SARS-CoV-2 Spike protein trimer (pink) bound to ACE2 (green). ( A ) Without glycans. ( B ) With N-glycans (red) identified using LC-MS on Spike and ACE2. ( C ) Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (‘S1-S2’, yellow) and S2’ (blue).

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Liquid Chromatography with Mass Spectroscopy, Glycoproteomics

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Full-length proteins expressed on cells include wild-type Spike-protein [v1] and human ACE2 [v2]. N-glycosylation sites are indicated by lollipop. Fc-his soluble proteins encode for S1-subunit [v3], RBD [v4] and soluble ACE2 [v5]. All constructs were co-expressed with fluorescent reporters separated by P2A. Note that the Fc-section also contains one N-glycosylation site. ( B ) Western blot for purified Fc-proteins from HEK293T probed with anti-Fc, anti-RBD or anti-ACE2 Ab. CD44-Fc is positive control. ( C ) Flow cytometry data showing S1-Fc (1.7 µg/mL) and RBD-Fc (0.35 µg/mL) binding to ACE2 expressed on HEK293T (middle panel). Spike expression enhances ACE2-Fc (1.4 µg/mL) binding (bottom). ( D ) Desialylation of Spike-protein expressed on 293 T/S had minimal effect on ACE2-Fc (0.7 µg/mL) binding. ACE2 desialylation on 293T/ACE2 increased binding of RBD-Fc (0.2 µg/mL) and S1-Fc (1.7 µg/mL) by 26–56% (paired experiments, *p<0.05). ( E ) Pseudovirus with DsRed-reporter were developed with three different envelope proteins. VSVG pseudotyped virus infected both HEK293T (black line) and stable 293T/ACE2 (red line) cells. Virus with Spike-WT and Spike-mutant entered 293T/ACE2 only. ( F ) Same titer of virus (0.3 µg/mL p24-equivalent) were treated with or without sialidase, prior to addition to stable 293T/ACE2 cells. Infection using Spike-mutant was higher compared to Spike-WT. Sialidase treatment of virus had no effect. ( G ) 293T/ACE2 cells were sialidase treated prior to addition of VSVG (0.3 µg/mL p24-equiv.), Spike-WT (1.5 µg/mL p24-equiv.) or Spike-mutant (0.2 µg/mL p24-equiv.) pseudovirus. Sialidase treatment did not affect viral entry. Abbreviations: Spike signal peptide (SP), N-terminal domain (NTD), receptor-binding domain (RBD), receptor-binding motif (RBM), subdomain 1 (SD1), subdomain 2 (SD2), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), heptad repeat 2 (HR2) transmembrane section (TM), cytoplasmic tail (CT), ACE2: Angiotensin-converting enzyme-2; VSVG: Vesicular stomatitis virus G-protein; WT: wild-type; mut: mutant.

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Glycoproteomics, Construct, Western Blot, Purification, Positive Control, Flow Cytometry, Binding Assay, Expressing, Virus, Infection, Mutagenesis

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Sialidase protocol validation. All lectins were directly conjugated with Alexa dyes. They were incubated with cells at 1–5 µg/mL for 15 min before a quick wash and cytometry measurement. Compared to untreated control (left), sialidase treatment (right) decreased SNA lectin binding to α2,6 sialylated structures by 15-fold and increased ECL binding to desialylated lactosamine chains (Galβ1,4GlcNAcβ) by an order of magnitude. ( B ) Pseudovirus assay. DsRed fluorescence in HEK293T and stable 293T/ACE2 cells upon addition of VSVG, Spike-WT and Spike-mutant pseudotyped virus. ( C ) Sialidase treatment of pseudovirus. % DsRed positive cell data are shown for study in (main manuscript). Viral entry was sialidase independent. ( D ) Sialidase treatment of HEK/ACE2 cells. Pseudovirus expressing VSVG, Spike-WT and Spike-mutant were added to cells under conditions described in (main manuscript). All error bars are standard deviations. Data are representative of 3 independent runs.

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Biomarker Discovery, Incubation, Cytometry, Control, Binding Assay, Fluorescence, Mutagenesis, Virus, Expressing

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Knocking out C1GALT1 and MGAT1 using CRISPR-Cas9 inhibits O- and N-glycan biosynthesis in HEK293Ts. ( B ) Sanger sequencing results of isogenic 293T clones shows indels on all 3 alleles of C1GALT1 (‘[O] - 293T’) and single allele of MGAT1 (‘[N] - 293T’) knockout cells. Wild-type (WT) sequence is on the first line. Lower line shows base deletions (hyphen) and insertions (black fonts) for individual KOs. sgRNA target sequence is in red and protospacer adjacent motif is underlined. ( C ) Increased VVA and reduced PHA-L binding confirm loss of O-linked glycans in [O] - 293Ts and N-glycans in [N] - 293Ts, respectively. ( D ) Knocking out N-glycans on Spike protein reduced ACE2-Fc binding in cytometry based binding studies. Knocking out Spike O-glycans increased ACE-2 binding. ( E–F ) Truncation of ACE2 N- and O-glycans did not affect either S1-Fc (panel E ) or RBD-Fc (panel F ) binding. ( G ) ACE2 was transiently expressed on 293T, [O] - 293T and [N] - 293 T cells. All pseudotyped virus efficiently entered ACE2 expressing cells. Virus was not titered for these runs, and thus comparison between viruses is not possible. *p<0.05 with respect to all other treatments. # p<0.05 with respect to 293T and [N] - 293T/ACE2 in panel G .

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: CRISPR, Glycoproteomics, Sequencing, Clone Assay, Knock-Out, Binding Assay, Cytometry, Virus, Expressing, Comparison

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Surface expression of Spike-protein and ACE2. Full-length Spike (top) and human ACE2 (bottom) were expressed in HEK 293T, [N] - 293T and [O] - 293T. Protein expression was measured in EGFP+ cells in the case of Spike (using anti-RBD), and on BFP+ cells in the case of ACE2 (using anti-ACE2), as these fluorescent reporters are co-expressed with surface proteins. Protein expression was comparable in all cells. Untransfected 293Ts serve as negative control. ( B ) Viral entry assay. Pseudovirus expressing VSVG envelope protein, Spike-WT or Spike-mutant were added to HEK 293 T cells transiently transfected to overexpress ACE2 (both wild-type 293T and glycosylation mutants). An additional control included 293 T cells not expressing ACE2, which only allowed entry of VSVG pseudotyped viral particles, but not Spike bearing virus. % cells that were DsRed (reporter) positive is shown at 72 hr. All treatments were statistically different except as indicated by n.s. (‘not significant’). All data are from of N > 3 repeats.

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Expressing, Negative Control, Mutagenesis, Transfection, Glycoproteomics, Control, Virus

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] - and [N] - 293 T cells. All nine viruses were applied at equal titer to stable 293T/ACE2. ( B–C ) O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. ( D ) Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. ( E ) Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] - 293T products due to truncation of glycan biosynthesis. ( F ) Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] - 293Ts is almost fully proteolyzed during viral production (red arrowhead). *p<0.05 with respect to all other treatments.

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Expressing, Mutagenesis, Produced, Glycoproteomics, Virus, Generated, Titration, Infection, Western Blot

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) VSVG, Spike-WT and Spike-mutant pseudovirus were produced in the presence of 15 µM kifunensine or vehicle control. The six viruses were added to 293T/ACE2 at equal titer. ( B–D ) Microscopy (panel B) and cytometry (panel C, D ) show ~90% loss of viral infection in the case of Spike-WT and Spike-mutant virus upon kifunensine treatment (*p<0.05). ( E ) Spike molecular mass is reduced in the western blots due to high-mannose glycan synthesis in runs with kifunensine. Intact Spike is reduced in the presence of kifunensine, in anti-FLAG blot. ( F ) The polybasic furin ‘RRAR’ site was substituted by a single ‘A’ amino acid in Spike-delta. Virus with Spike-delta were expressed both in the presence of vehicle and kifunensine. Western blot shows lack of S1-S2 cleavage in this construct. In viral entry assay, kifunensine reduced DsRed expression in 293T/ACE2 cells, even in the case of Spike-delta pseudovirus (*p<0.05). Similar observation was made at two different viral titers (0.3 and 0.6 μg/mL p24 equivalent).

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Mutagenesis, Produced, Control, Microscopy, Cytometry, Infection, Virus, Western Blot, Glycoproteomics, Construct, Expressing

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293 T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. ( B ) SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral entry ( B ) assay are listed. ( C ) Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Binding Assay, Expressing, Generated, Virus

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Binding Assay, Plasmid Preparation, Recombinant, Knock-Out, Derivative Assay

Journal: mBio

Article Title: SARS-CoV-2 infection alters mitochondrial and cytoskeletal function in human respiratory epithelial cells mediated by expression of spike protein

doi: 10.1128/mbio.00820-23

Figure Lengend Snippet: SCV2 pseudovirus induces SCV2-specified gene expression in NHBE. NHBE cells were infected with SCV2 spike or control pseudovirus for 72 h, and ( A ) the infection efficiency was imaged by DsRed fluorescent signal (32 ×, scale bars of 100 µm). Gene expression of ( B ) HES1 and ( C ) KLF2 was examined by qPCR and normalized by housekeeping gene GAPDH. The error bars represent ±standard error of the mean (SEM) ( n = 3). Statistics were determined by Welch’s test, with P < 0.05 considered statistically significant. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HES1, hes family BHLH transcription factor 1; KLF2, KLF transcription factor 2; NHBE, normal human bronchial epithelial; Spike, SARS-CoV-2 spike glycoprotein; VSV-G, VSV glycoprotein.

Article Snippet: Full-length VSV glycoprotein (VSV-G) (Addgene, #12259) and pRP-Neo-EF1A-SARS-Cov2-Spike delta 18, fused with a DsRed tag, were kindly gifted from Vasudevan, Anand’s lab. Plasmid for the SARS-CoV-2 spike delta 18 glycoprotein has a C-terminal deletion of the last 18 amino acids to improve binding to ACE2.

Techniques: Gene Expression, Infection, Control

Journal: mBio

Article Title: SARS-CoV-2 infection alters mitochondrial and cytoskeletal function in human respiratory epithelial cells mediated by expression of spike protein

doi: 10.1128/mbio.00820-23

Figure Lengend Snippet: SCV2 infection enhances metabolism in the host cells. Using KEGG pathway analysis, differentially expressed genes involved in ( A ) nucleotide, ( B ) amino acid, ( C ) fatty acid, and carbohydrate metabolisms were shown in IAV/mock and SCV2/mock at 24 hpi. After 72 h of pseudovirus infection in NHBE cells, gene expression of ( E ) glycolysis rate limiting PKLR, ( F ) SIRT3, and ( G ) mitochondrial marker TOMM22 was measured by qPCR and normalized by housekeeping gene GAPDH (5–9 inserts from two donors), and ( H ) the mitochondrial localization was observed by TOMM22 immunofluorescent staining. The immunofluorescence images were taken at 63× oil objective, scale bars of 20 µm. The error bars represent ±standard error of the mean (SEM) (two donors). Statistics were determined by Welch’s test, with P < 0.05 considered statistically significant. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. PKLR, pyruvate kinase L/R; SIRT3, NAD+-dependent protein deacetylases 3; TOMM22, translocase of the outer membrane of mitochondria; Spike, SARS-CoV-2 spike glycoprotein; VSV-G, VSV glycoprotein.

Article Snippet: Full-length VSV glycoprotein (VSV-G) (Addgene, #12259) and pRP-Neo-EF1A-SARS-Cov2-Spike delta 18, fused with a DsRed tag, were kindly gifted from Vasudevan, Anand’s lab. Plasmid for the SARS-CoV-2 spike delta 18 glycoprotein has a C-terminal deletion of the last 18 amino acids to improve binding to ACE2.

Techniques: Infection, Gene Expression, Marker, Staining, Immunofluorescence, Membrane

Journal: mBio

Article Title: SARS-CoV-2 infection alters mitochondrial and cytoskeletal function in human respiratory epithelial cells mediated by expression of spike protein

doi: 10.1128/mbio.00820-23

Figure Lengend Snippet: SCV2 spike protein infection impairs metabolism in human epithelial cells. ( A ) The pseudovirus infection efficiency of Calu3 cells after 48 h post infection was examined by DsRed fluorescent signal (32×, scale bars of 100 µm). ( B ) ECAR and ( C ) OCR were examined in the pseudovirus-infected Calu3 cells. ( D ) Basal respiration, ( E ) maximal respiration, and ( F ) ATP-linked respiration were shown. Gene expression of ( G ) SIRT3 and ( H ) TOMM22 as well as ( I ) immunofluorescence of TOMM22 (green) and Hoechst (blue) staining were taken at 63× oil objective, scale bar of 20 µm. The error bars represent ±standard error of the mean (SEM) (4–10 inserts/group). Statistics were determined by Welch’s test, with P < 0.05 considered statistically significant. ECAR, extracellular acidification rate; OCR, oxygen consumption rate; RHOB, ras homolog family member B; SIRT3, NAD+-dependent protein deacetylases 3; TOMM22, translocase of the outer membrane of mitochondria; Spike, SARS-CoV-2 spike glycoprotein; VSV-G, VSV glycoprotein.

Article Snippet: Full-length VSV glycoprotein (VSV-G) (Addgene, #12259) and pRP-Neo-EF1A-SARS-Cov2-Spike delta 18, fused with a DsRed tag, were kindly gifted from Vasudevan, Anand’s lab. Plasmid for the SARS-CoV-2 spike delta 18 glycoprotein has a C-terminal deletion of the last 18 amino acids to improve binding to ACE2.

Techniques: Infection, Gene Expression, Immunofluorescence, Staining, Membrane

Journal: mBio

Article Title: SARS-CoV-2 infection alters mitochondrial and cytoskeletal function in human respiratory epithelial cells mediated by expression of spike protein

doi: 10.1128/mbio.00820-23

Figure Lengend Snippet: SCV2 infection disrupts cell-cell adhesion in the human epithelium. Using KEGG pathway analysis, differential expression of genes involved in ( A ) tight junction, ( B ) cell adhesion, ( C ) focal adhesion, and ( D ) regulation of actin cytoskeleton was shown in IAV/mock and SCV2/mock at 24 hpi. After 72 h of the pseudovirus infection in NHBE cells, expressions of ( E ) CFL1 transcript, ( F ) CFL1 protein, and ( G ) RHOB transcript were examined ( n = 3–12). ( G ) Immunofluorescence of TOMM22 (green) and Hoechst (blue) staining was taken at 63× oil objective, scale bar of 10 µm. TEER was measured in the pseudovirus-infected ( H ) NHBE cells and ( I ) Calu3 cells ( n = 8). The error bars represent ±standard error of the mean (SEM). Statistics were determined by Welch’s test, with P < 0.05 considered statistically significant. CFL1, cofilin-1; ECAR, extracellular acidification rate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OCR, oxygen consumption rate; TEER, transepithelial electrical resistance; TOMM22, translocase of the outer membrane of mitochondria; Spike, SARS-CoV-2 spike glycoprotein; VSV-G, VSV glycoprotein.

Article Snippet: Full-length VSV glycoprotein (VSV-G) (Addgene, #12259) and pRP-Neo-EF1A-SARS-Cov2-Spike delta 18, fused with a DsRed tag, were kindly gifted from Vasudevan, Anand’s lab. Plasmid for the SARS-CoV-2 spike delta 18 glycoprotein has a C-terminal deletion of the last 18 amino acids to improve binding to ACE2.

Techniques: Infection, Quantitative Proteomics, Immunofluorescence, Staining, Membrane

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative double immunofluorescence staining of ACE2 and endothelial cell marker CD31 in the blood vessels of human nasal turbinates using six different anti-ACE2 antibodies and anti-CD31. b Double immunofluorescence staining of ACE2 and type II pneumocyte marker mucin 1 (MUC1) in the human lung using six different anti-ACE2 antibodies and anti-MUC1. Abcam ab15348 clone yielded the most robust staining of pneumocytes, while the other clones showed negligible or less specific membrane staining. Scale bars: 20 μm (top) and 5 μm (bottom).

Article Snippet: For transient transfection of IMCD3 cells, IMCD3 cells were grown to ~80% confluence and transfected with 5 μg of full-length ACE2 plasmid DNA/1 × 10 6 cells using Fugene6 (Promega).

Techniques: Double Immunofluorescence Staining, Marker, Staining, Clone Assay, Membrane

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: Representative images of human tissues on a tissue microarray (TMA) stained by chromogenic immunohistochemistry using antibodies targeting the ACE2 protein (brown) and counterstained with hematoxylin (blue). Highest ACE2 expression was observed in the villi of the intestinal tract (jejunum), renal tubules, testis, and glandular cells in the seminal vesicle. Minimal to no/non-specific staining can be seen in the heart, stomach, spleen, skin, and liver. Staining of lung pneumocytes was observed using Abcam ab15348, and less specifically with Sigma HPA000288 (Fig. 2b; Supplementary Table 1 ). Scale bars: 100 μm.

Article Snippet: For transient transfection of IMCD3 cells, IMCD3 cells were grown to ~80% confluence and transfected with 5 μg of full-length ACE2 plasmid DNA/1 × 10 6 cells using Fugene6 (Promega).

Techniques: Microarray, Staining, Immunohistochemistry, Expressing

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative double immunofluorescence staining of ACE2 and acetylated α-tubulin (ACTUB) on normal human nasal turbinate, ethmoid sinus, uncinate process (sinus), trachea, and bronchus, using anti-ACE2 and anti-ACTUB antibodies, respectively. b Representative double immunofluorescence staining of ACE2 and ACTUB on normal C57BL/6J mouse nasal turbinate and trachea. c Immunofluorescent staining of (top panel) ACE2, cilia marker ADP-ribosylation factor-like protein 13B (ARL13B), and cilia centrosome marker FGFR1 oncogene partner (FOP); (bottom panel) ACE2, and cilia markers ACTUB and ARL13B in a ciliated mouse cell line, IMCD3. d Immunofluorescent staining of ACE2 in the primary cilia of IMCD3 cells transiently transfected with human ACE2 (yellow outline) compared to endogenous mouse ACE2 (blue outline). e Quantified percentages of endogenous ACE2-positive cilia (34.67 ± 13.58%; control (Ctrl)) versus cilia with overexpressed human ACE2 (82.67 ± 4.73%). Ciliated cells were identified by staining of ARL13B. Error bars represent mean ± SD. ( n = 100 cells examined per experiment over three independent experiments). (Two-tailed Student’s t test, ** p = 0.004). f Representative multiplexed images of in situ hybridization against the SARS-CoV-2 Spike mRNA, in combination with immunofluorescence staining of ACE2 and the differentiated epithelial cell marker cytokeratin 8 (KRT8). SARS-CoV-2 Spike mRNA expression (red) was detected within ciliated epithelial cells containing motile cilia positive for ACE2 (green). The nuclei were stained using DAPI (blue) as a counterstain. Scale bars: 20 μm ( a , b top panels; f large panels); 5 μm ( a , b bottom panels; f small panels); 2 μm ( c , d ).

Article Snippet: For transient transfection of IMCD3 cells, IMCD3 cells were grown to ~80% confluence and transfected with 5 μg of full-length ACE2 plasmid DNA/1 × 10 6 cells using Fugene6 (Promega).

Techniques: Double Immunofluorescence Staining, Staining, Marker, Transfection, Control, Two Tailed Test, In Situ Hybridization, Immunofluorescence, Expressing

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative immunofluorescence double staining of ACE2 and mucin 5AC (MUC5AC) reveals absence of co-localization of ACE2 within secretory goblet cells in the human nasal turbinate, uncinate process, and bronchus. b Representative in situ hybridization using an ACE2 probe in combination with an anti-MUC5AC antibody. ACE2 mRNA expression (red dots) was not detected within goblet cells marked by MUC5AC in the nasal turbinate, uncinate process, and trachea. Nuclei were stained using DAPI. Scale bars: 20 μm (top) and 5 μm (bottom).

Article Snippet: For transient transfection of IMCD3 cells, IMCD3 cells were grown to ~80% confluence and transfected with 5 μg of full-length ACE2 plasmid DNA/1 × 10 6 cells using Fugene6 (Promega).

Techniques: Immunofluorescence, Double Staining, In Situ Hybridization, Expressing, Staining

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a No statistically significant changes in ACE2 expression was detected among patients less than or greater than 65 years of age, males versus females, and patients with varying smoking history. (Two-tailed Mann–Whitney test or Kruskal–Wallis test, p > 0.05). b No statistically significant difference in ACE2 expression was observed between healthy controls and patients with chronic rhinosinusitis with polyps (CRSwNP) or without polyps (CRSsNP). (Kruskal–Wallis test, p > 0.05). c No statistically significant difference in ACE2 expression was noted between distinct human nasal tissue sites/regions. (Two-tailed Mann–Whitney test or Kruskal–Wallis test, p > 0.05). UNC uncinate process, Turb nasal turbinates, Eth ethmoid sinus, NP benign nasal polyps. The bottom and top of the box plots represent the 25th and 75th percentiles, respectively. The bands within the box show the median value, and the whiskers extending from both ends of the boxes are minimum and maximum values. Each dot represents one patient.

Article Snippet: For transient transfection of IMCD3 cells, IMCD3 cells were grown to ~80% confluence and transfected with 5 μg of full-length ACE2 plasmid DNA/1 × 10 6 cells using Fugene6 (Promega).

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Quantification of ACE2 in controls and patients taking ARBs and ACEI. In the Stanford cohort, ACE2 is slightly but statistically significantly lower in patients taking ACEI (0.19 ± 0.02) compared to controls (0.26 ± 0.06). (Kruskal–Wallis test p = 0.021; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.043). There were no statistically significant differences in ACE2 expression between patients taking ARBs and controls in the Stanford, National Taiwan University Hospital (NTUH), and China Medical University Hospital (CMUH) cohorts. b In the Stanford cohort, when including only controls with hypertension (HTN) on other medications (“HTN w/o ARBs/ACEI”), ACE2 expression was statistically different between the groups (Kruskal–Wallis test, p = 0.044) but Dunn’s multiple comparison post-hoc test did not reveal any statistical significance between the three groups. No statistically significant differences were seen among patients taking ARBs compared to controls. c When cohorts from all three institutions were normalized by Z -score and integrated, patients taking ACEI (−0.72 ± 0.42) had a lower ACE2 expression compared to controls with hypertension (0.41 ± 1.07). (Kruskal–Wallis test, p = 0.032; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.043). Patients taking ARBs (−0.15 ± 0.95) showed a trend towards lower ACE2 compared to controls with hypertension, but this was not statistically significant. d ACE2 expression among patients of older (≥65 years) and younger (<65 years) age taking ARBs or ACEI was not statistically divergent from control patients of the same age group. (Kruskal–Wallis test, p > 0.05). e ACE2 expression among male and female patients on ARBs or ACEI trended comparably or lower than same-sex controls except for males taking ARBs in the CMUH group who showed a trend towards higher ACE2 expression. No statistically significant differences were observed. (Kruskal–Wallis test, p > 0.05). f Among non-smokers, there was a statistically significant trend towards lower ACE2 expression in patients taking ACEI compared to controls in the Stanford group (Kruskal–Wallis test, p = 0.021; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.035). No statistical significance was observed with the non-smokers on ARBs. All data are noted as mean ± SD. Kruskal–Wallis test was used for three group comparisons and two-tailed Mann–Whitney test was used for two-group comparisons. Box plots are similar in format to Fig. .

Article Snippet: For transient transfection of IMCD3 cells, IMCD3 cells were grown to ~80% confluence and transfected with 5 μg of full-length ACE2 plasmid DNA/1 × 10 6 cells using Fugene6 (Promega).

Techniques: Comparison, Expressing, Medications, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: The luminal differentiated airway epithelial cells consist of ciliated columnar cells (~80%) and secretory goblet cells (~20%). Club cells are infrequently found in the human upper airway. The basal cell layer, which faces the lamina propria, is comprised of both basal and suprabasal cells, which are considered multipotent progenitors capable of renewing the airway epithelium. This schematic depicts how SARS-CoV-2 may bind to ACE2 expressed on the cilia of the nasal cavity following exposure to respiratory droplets or airborne particles.

Article Snippet: For transient transfection of IMCD3 cells, IMCD3 cells were grown to ~80% confluence and transfected with 5 μg of full-length ACE2 plasmid DNA/1 × 10 6 cells using Fugene6 (Promega).

Techniques: