Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 6. Surface expression and soluble receptor binding of wild-type and mutant SARS-CoV S 19 proteins. (A) Cell surface expression of the wild-type or mutant SARS-CoV S 19 protein was measured by flow cytometry. (B and C) Binding of the soluble hACE2-Fc receptor protein by cells expressing wild-type or mutant SARS-CoV S 19 detached with trypsin-EDTA (B) or EDTA alone (C). (D to F) Representative flow cytometry overlays shown to the right of corresponding bar graphs. Mock-transfected cells are shown in dark gray, and SARS-CoV S 19 is shown by a black line; representative SARS-CoV S 19 mutant proteins are shown by color (SARS-CoV S 19 Y1188A, green; S 19 W1194A, red; S 19 Y1197A, orange; S 19 Y1188A/Y1191A, cyan; and S 19 W1194/W1199A, yellow, respectively). Error bars represent standard errors of percent wild-type levels between at least three independent experiments.

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Expressing, Binding Assay, Mutagenesis, Cytometry, Transfection

Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 8. Surface expression of hACE2. HEK-293 cells stably ex- pressing high levels of hACE2 were selected by flow cytometry.

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Expressing, Stable Transfection, Cytometry

Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 9. Alanine substitutions in the JMD of the SARS-CoV S 19 protein reduce receptor-dependent entry of pseudotyped viruses. Rel- ative transduction activity of pseudotyped viruses with wild-type or mutant SARS-CoV S 19 proteins is shown. Pseudotypes in filtered medium were used to transduce HEK-293/hACE2 cells. Beta-galacto- sidase expression was analyzed 2 days posttransduction. Transduction units per ml were calculated {TU/ml [(% cells expressing reporter/ 100) total number of cells per well]/ml inoculum}. Data are repre- sented as percent transduction activity of wild-type SARS-CoV S 19 proteins and are representative of three to six experiments. Error bars represent standard errors. Student’s t test was used to evaluate the significance of differences between the transducing activities of pseudotyped viruses with mutant or wild-type proteins. (*, P 0.01 to 0.05; **, P 0.01 to 0.001; ***, P 0.0001).

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Transduction, Activity Assay, Mutagenesis, Expressing

Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 10. Trypsin enhances receptor-dependent cell-cell fusion induced by wild-type SARS-CoV S 19 protein. The time course of syncytium formation was determined after 30 min, 1 h, 2 h, or 3 h. HEK-293 cells transfected with cDNA encoding the wild-type SARS-CoV S 19 glycoprotein (green) were stained with Celltracker Green (Molecular Probes) and were detached from the monolayer with EDTA alone or trypsin-EDTA and added to a 50% confluent monolayer of HEK-293/hACE2 cells (blue) stained with Hoechst 33342 (Sigma) at a ratio of one S-expressing cell to three hACE2-expressing cells. Cells were fixed with paraformaldehyde 2 h postmixing. Pictures were taken with a 20 objective. Pictures are representative of two independent experiments..

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Transfection, Staining, Expressing

Journal: Journal of Virology

Article Title: Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

doi: 10.1128/jvi.01805-07

Figure Lengend Snippet: FIG. 11. JMD mutations reduce receptor-dependent cell-cell fu- sion induced by SARS S 19 proteins. (A) Fusion of HEK-293 cells expressing wild-type or mutant SARS-CoV S 19 proteins or empty vector with HEK-293/hACE2 cells. Fusion was scored as the percent- age of nuclei present in syncytia of any size at 2 h after mixing cells expressing spike and receptor. Background levels of fusion up to 15% were seen in HEK-293 cells. Data are expressed as a percentage of wild-type SARS-CoV S 19 fusion with background levels of fusion subtracted. Data are representative of three independent experiments. P values are 0.02 for all mutant proteins relative to results for wild- type SARS-CoV S 19. (B and C) Analysis of syncytium size induced by tyrosine-to-alanine (B) or tryptophan-to-alanine (C) mutants of the SARS-CoV S 19 protein. Each individual syncytium was scored for size (number of nuclei). Results are plotted as the cumulative percent- age of cells containing a given number of nuclei or less.

Article Snippet: HEK-293 cells stably expressing hACE2 were made by transfection of HEK-293 cells with pcDNA3.1 hACE2, selection with G418 (Invitrogen), and sorting for hACE2 expression with goat anti-human hACE2 AF933 (R&D Systems, Minneapolis, MN) and phycoerythrin (PE)-conjugated anti-goat immunoglobulin G (IgG) (Jackson Immunoresearch, West Grove, PA) antibodies.

Techniques: Expressing, Mutagenesis, Plasmid Preparation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Lung-Targeted Lipid Nanoparticle-Delivered siUSP33 Attenuates SARS-CoV-2 Replication and Virulence by Promoting Envelope Degradation.

doi: 10.1002/advs.202406211

Figure Lengend Snippet: Figure 6. LNP-siUSP33 reduces viral load and lung pathology in vivo. A) C57BL/6 mice were nasally infected with AAV-hACE2 and injected with indicated reagents via retro-orbital. On the second day of injection, the mice were infected nasally with 1 × 105 FFU WT SARS-CoV-2. All the lung tissues were collected on the 8th day after infection. B) The body weights of the mice were recorded daily for 8 days after infection. Quantification of body weights compared to pre-infection was shown as mean ± SD (n = 5 independent experiments). C) The lung tissues of mice were collected on the 8th day after infection and then lyzed to detect the levels of indicated proteins via immunoblotting. D) Total RNA was extracted from the lung tissues, and the relative levels of SARS-CoV-2 N genes were detected. E) The lungs were ground into homogenates and then infected Vero cells, and viral titers were calculated using the FFA. F) Immunohistochemistry analyses with anti-SARS-CoV-2 N antibody was performed to assess the relative amount of SARS- CoV-2 in the lungs. Scale bar, 100 μm. G) H&E staining was performed to observe the intensity of the inflammatory infiltrate. Scale bars, 1000 μm (up) and 50 μm (down). H) Total RNA was extracted from the lungs and relative levels of indicated inflammatory genes were detected using qRT-PCR. I) Schematic diagram of the K18-hACE2 mice of SARS-CoV-2 infection. J) Total RNA was extracted from the lung tissues collected on day 4 after infection, and the relative levels of SARS-CoV-2 N genes were detected. K–M) The viral loads of the lungs (K), livers (L), and kidneys (M) collected on day 4 were calculated using FFA. Student’s t test (unpaired, two-tailed) was used to compare two independent groups, and a two-way ANOVA test was performed for comparisons of multiple groups. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Article Snippet: All siRNAs were synthesized by RIBOBIO and the sense strand sequences are shown below. hUSP33#1: CCCAGUAAUACAACAUUAATT hUSP33#2: GGAGAAUAGAUGUUCAUAUTT hUSP33#3: GCUGCAUUCAUCAAGUCAUTT mUSP33#1: GCAGGAGACAAAGCAUUAUTT mUSP33#2: GCCGGCUAAUCUGUUCCAATT mUSP33#3: GCAGAGCCUCAGAAUCUAUTT mUSP33#4: GCUGAACCUGGCCCUAUUUTT Antibodies and Other Reagents: The following antibodies and reagents were used for immunoblotting and immunoprecipitation in this study: Rabbit anti-Flag monoclonal antibody (CST, 14 793); Rabbit anti-HA monoclonal antibody (CST, 3724); Mouse anti-Strep-Tag II monoclonal antibody (Abbkine, 8C12); Rabbit anti-GST monoclonal antibody (CST, 2625); Mouse anti-His monoclonal antibody (CST, 2366); Rabbit anti-Myc monoclonal antibody (CST, 13 987); Mouse anti-β-actin monoclonal antibody (TransGen Biotech, HC201-01); Rabbit anti-α-Tubulin polyclonal antibody (Proteintech, 11224-AP); Rabbit anti-Histone-H3 polyclonal antibody (Proteintech, 17168-1AP); Mouse anti-Ubiquitin monoclonal antibody (SantaCruz, sc-8017); Rabbit anti-USP33 polyclonal antibody (Proteintech, 20445-1-AP); Rabbit anti-RNF5 monoclonal antibody (ab308066); Rabbit anti-SARS-CoV-2 E monoclonal antibody (abcam, ab308371); Rabbit anti-SARS-CoV-2-M monoclonal antibody (abcam, ab308415); Rabbit anti-SARS-CoV-2-ORF7b monoclonal antibody (abcam, ab313933); Mouse anti-SARS-CoV-2-N monoclonal antibody (SinoBiological, 40143- MM05); Rabbit anti-SARS-CoV-2-S polyclonal antibody (SinoBiological, 40590-T46); Rabbit anti-SARS-CoV-2 NSP3 monoclonal antibody (CST, 88 086); Rabbit anti-hACE2 polyclonal antibody (CST, 4355); Goat antiRabbit and anti-Mouse HRP Conjugated secondary antibodies (TransGen Biotech); Goat anti-Rabbit Alexa Fluo 488 Conjugated secondary antibodies (Invitrogen, A-11008); Goat anti-Mouse Alexa Fluo 555 Conjugated secondary antibodies (Invitrogen, A-21422); DAPI (Beyotime, C1002); AntiFlag magnetic Beads (Sigma, M8823); Anti-HA magnetic beads (Sigma, SAE0197); Anti-Myc magnetic beads (Sigma, SAE0201); Protein A/G agarose (GE healthcare); Magnetic Beads-conjugated Rabbit anti GST-Tag mAb (ABclonal, AE122); Reduced Glutathione (Solarbio, G8180) and IPTG (Solarbio, I1020).

Techniques: In Vivo, Infection, Injection, Western Blot, Immunohistochemistry, Staining, Quantitative RT-PCR, Two Tailed Test

Journal: Science Bulletin

Article Title: Protoporphyrin IX and verteporfin potently inhibit SARS-CoV-2 infection in vitro and in a mouse model expressing human ACE2

doi: 10.1016/j.scib.2020.12.005

Figure Lengend Snippet: Protoporphyrin IX and verteporfin bind human ACE2 protein. (a) Structures of protoporphyrin IX and verteporfin. (b) Docking of ACE2 peptidase domain (PD) with protoporphyrin IX (blue) and verteporfin (pink). The 3D structure of PD is from the cryo-electron microscopy structure of the ACE2-B 0 AT1 complex (PDB ID: 6 m18). The surface of PD is shown. (c) Interactions of protoporphyrin IX (upper) or verteporfin (bottom) with ACE2 residues. (d) Binding profiles of protoporphyrin IX or verteporfin to ACE2-Fc protein measured with BLI assay.

Article Snippet: To detect hACE2 expression, the sections were first incubated in blocking reagent and then with hACE2 antibody (1:100 dilution, Proteintech) at 4 °C overnight, followed by incubation with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000 dilution, Invitrogen).

Techniques: Cryo-Electron Microscopy, Binding Assay

Journal: Science Bulletin

Article Title: Protoporphyrin IX and verteporfin potently inhibit SARS-CoV-2 infection in vitro and in a mouse model expressing human ACE2

doi: 10.1016/j.scib.2020.12.005

Figure Lengend Snippet: Effective inhibition of SARS-CoV-2 infection by protoporphyrin IX and verteporfin in SARS-CoV-2-infected hACE2 mice. (a) Schematic representation of the experiment timeline. (b) Relative viral RNA levels in lung tissues from each group. Data are relative to that of the DMSO-treated group and statistical significance was calculated using unpaired two-tailed t -test. The data of each group were compared with those of DMSO group, respectively. ** P < 0.01 and *** P < 0.001. (c) Immunohistochemical staining of hACE2 and viral N protein in lung tissue samples from each group. (d) Representative H & E staining of lung tissue sections from each group.

Article Snippet: To detect hACE2 expression, the sections were first incubated in blocking reagent and then with hACE2 antibody (1:100 dilution, Proteintech) at 4 °C overnight, followed by incubation with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000 dilution, Invitrogen).

Techniques: Inhibition, Infection, Two Tailed Test, Immunohistochemical staining, Staining

Journal: PLOS Biology

Article Title: Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection

doi: 10.1371/journal.pbio.3001989

Figure Lengend Snippet: ( A) Generation of the hACE2 fl/y and hACE2 hypo alleles using gene targeting of the mouse Ace2 locus. ( B) Immunoblotting of whole tissue lysates from WT and hACE2 fl/y organs using an antibody recognizing both mouse and human ACE2 (anti-panACE2). Total protein stain used to assess protein input. Representative of 3 independent experiments, n = 3 animals per genotype. ( C) qPCR was performed using primers that detect untranslated 5′ mRNA sequences present in WT Ace2 , hACE2 fl/y , and hACE2 hypo/y alleles using the indicated tissues. ( D) Immunoblotting of whole tissue lysates from WT, hACE2 fl/y , hACE2 hypo/y , K18-hACE2 transgenic, and hACE2 knock-in mice generated by the Wentworth lab ( hACE2 Wentworth/y ) was performed using anti-panACE2 and anti-β-actin (as a loading control). Representative of 3 independent experiments, n = 3 animals per genotype. ( E) Immunoblotting of whole brain lysates from humans (Human) and the indicated mouse strains defined in (D) was performed using anti-panACE2 and b-actin antibodies. Representative of 3 independent experiments, n = 3 animals per genotype. ( F, G) Immunoblotting of whole tissue lysates from WT, hACE2 fl/y , and hACE2 del/y mice was performed using antibodies that specifically recognize hACE2 protein or anti-panACE2 antibodies. Β-actin immunoblotting is shown as a loading control. Each blot is representative of 4 independent experiments, n = 4 animals per genotype. ( H) Immunohistochemistry of ACE2 using hACE2 antibodies and neurons using NeuN or β3-tubulin antibodies in WT and hACE2 fl/y mouse cerebral cortex. Arrows indicate NeuN or β3-tubulin-positive neurons colocalized with ACE2 staining. Representative of n = 3 per genotype. Scale bars 50 μm. ( I) Immunohistochemistry using pan-ACE2 and E-Cadherin antibodies is shown for WT and hACE2 fl/y epithelium from the anterior and posterior part of the nasal cavity. Arrowheads indicate ACE2+ epithelial cells. n = 3 animals per genotype. ( J) Immunohistochemistry using pan-ACE2 and hACE2 antibodies is shown for WT and hACE2 fl/y OE costained with OMP (mature olfactory sensory neurons) or epithelial cadherin (E-cadherin, sustentacular cells). Arrowheads indicate ACE2+ olfactory epithelial cells. n = 3 animals per genotype. Scale bars 50 μm. ( K) Immunostaining using pan-ACE2 antibodies is shown for WT and hACE2 fl/y lung costained with antibodies recognizing DC-LAMP. Arrowheads indicate ACE2+ AT2 cells. Arrows indicate ACE2+ bronchial epithelial cells. n = 3 animals per genotype. Scale bars 50 μm. ( L) Immunostaining using pan-ACE2 and hACE2 antibodies is shown for WT and hACE2 fl/y kidney costained with E-cadherin (epithelium). Arrowheads indicate ACE2+ tubular epithelial cells. n = 3 animals per genotype. Scale bars 50 μm. (M) Immunostaining using pan-ACE2 and hACE2 antibodies is shown for WT and hACE2 fl/y small intestine costained with E-cadherin (enterocytes). Arrowheads indicate ACE2+ enterocytes. N = 3 animals per genotype. Scale bars 50 μm. ns p > 0.05; * p < 0.05; ** p < 0.01; **** p < 0.0001 by unpaired, two-tailed t test. Numerical data in corresponding tab. hACE2, human ACE2; OE, olfactory epithelium; OMP, olfactory marker protein; WT, wild-type.

Article Snippet: Primary antibodies used for immunoblotting: pan-ACE2 (1:1,000; R&D Systems; AF933), hACE2 (1:1,000; Atlas Antibodies; AMAB91259), β-Actin (1:1,000; Cell Signaling; 4970).

Techniques: Western Blot, Staining, Transgenic Assay, Knock-In, Generated, Control, Immunohistochemistry, Immunostaining, Two Tailed Test, Marker

Journal: PLOS Biology

Article Title: Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection

doi: 10.1371/journal.pbio.3001989

Figure Lengend Snippet: ( A) Experimental design for acute infection with SARS-CoV-2 virus. ( B, C) Weight loss and survival of hACE2 fl/y and hACE2 del/y mice after infection with 10 5 viral titer per mouse. n = 8 for both genotypes, two independent experiments. ( D, E) Weight loss and survival of hACE2 fl/y and hACE2 hypo/y mice after infection with 10 5 viral titer per mouse. n = 8 for both genotypes, two independent experiments. ( F, G) Weight loss and survival of hACE2 fl/y and hACE2 Wentworth/y mice after infection with 10 5 PFU of SARS-CoV-2 virus. n = 8 ( hACE2 fl/y ) and 5 ( hACE2 Wentworth/y ), one experiment. ( H, I) Weight loss and survival of littermate male and female hACE2 fl/y mice after infection with 10 4 titer of SARS-CoV-2 virus. n = 11 (male) and 6 (female), one experiment. ( J, K) Weight loss and survival of littermate male hACE2 fl/y mice after infection with 10 5 titer of the USA-WA1 or Omicron BA.1 strains of SARS-CoV-2 virus. n = 10 (USA-WA1) and 7 (Omicron BA.1), two experiments. Note: Data for hACE2 fl/y infections at 10 5 titer are reused throughout panels (B-E) in this figure as littermate controls were not applicable and infections done contemporaneously with the same viral stock. ns p > 0.05; * p < 0.5; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by multiple unpaired two-tailed t test or log-rank Mantel Cox test. Numerical data in corresponding tab. DPI, days postinfection; hACE2, human ACE2; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.

Article Snippet: Primary antibodies used for immunoblotting: pan-ACE2 (1:1,000; R&D Systems; AF933), hACE2 (1:1,000; Atlas Antibodies; AMAB91259), β-Actin (1:1,000; Cell Signaling; 4970).

Techniques: Infection, Virus, Two Tailed Test

Journal: PLOS Biology

Article Title: Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection

doi: 10.1371/journal.pbio.3001989

Figure Lengend Snippet: ( A) Immunodetection of ACE2 using pan-ACE2 antibodies and DC-LAMP in wild-type hACE2 fl/y , and Sfptc CreERT2/+ ; hACE2 fl/y mouse lungs 6 days after infection with 10 4 PFU of SARS-CoV-2. Arrowheads indicate DC-LAMP+ AT2 cells. n = 4 for both genotypes, one experiment. ( B) Immunohistochemistry of hACE2 fl/y and Sfptc CreERT2/+ ; hACE2 fl/y mouse lungs 6 days after infection with 10 4 PFU of SARS-CoV-2 virus was performed using antibodies that recognize SARS-CoV-2 nucleocapsid, DC-LAMP (AT2 cells), and PDPN (AT1 cells) as well as the nuclear stain DAPI. Arrows indicate nucleocapsid colocalized with PDPN. N = 4 for both genotypes, one experiment. ( C) qPCR was performed on hACE2 fl/y and Sfptc CreERT2/+ ; hACE2 fl/y mouse lungs harvested 6 days after infection with 10 4 PFU of SARS-CoV-2 to measure total viral load. N = 4 for both genotypes, one experiment. ( D) Immunohistochemistry of hACE2 fl/y and Hopx CreERT2/+ ; hACE2 fl/y mouse lungs 2 days after infection with 10 5 PFU of SARS-CoV-2 virus was performed using antibodies that recognize SARS-CoV-2 nucleocapsid, DC-LAMP, and PDPN as well as the nuclear stain DAPI. Arrows indicate nucleocapsid colocalized with PDPN in AT1 cells. Arrowheads indicate nucleocapsid colocalized with DC-LAMP in AT2 cells. The boxed regions show DC-LAMP staining (red) only in the indicated nucleocapsid positive (green) AT2 cells because the dim DC-LAMP signal is obscured by nucleocapsid signal when both are visible. N = 4–5 for both genotypes, one experiment. ( E) qPCR was performed on hACE2 fl/y and Hopx CreERT2/+ ; hACE2 fl/y mouse lungs harvested 2 days after infection with 10 5 PFU of SARS-CoV-2 to measure total viral load. N = 4–6 for both genotypes, one experiment. ( F) Immunohistochemistry of hACE2 fl/y and Shh Cre/+ ; hACE2 fl/y mouse lungs 2 and 6 days after infection with 10 4 or 10 5 PFU of SARS-CoV-2 virus was performed using antibodies that recognize SARS-CoV-2 nucleocapsid, DC-LAMP (AT2 cells), and PDPN (AT1 cells) as well as the nuclear stain DAPI. N = 6 for all genotypes, two independent experiments. Arrows indicate nucleocapsid colocalized with PDPN. ( G) qPCR was performed on hACE2 fl/y and Shh Cre/+ ; hACE2 fl/y mouse lungs harvested 2 and 6 days after infection with 10 4 or 10 5 PFU of SARS-CoV-2 to measure total viral RNA load. Simultaneous measurement using whole blood was performed 6 days after infection (shown in red) to measure circulating levels. ( H) Infectious viral load was measured by PFU from hACE2 fl/y and Shh Cre/+ ; hACE2 fl/y mouse lungs 6 days after infection with 10 5 PFU of SARS-CoV-2. N > 6 for both genotypes. Note: hACE2 fl/y 10 4 titer data for 3C and 3G are the same. Scale bars in all images 50 μm. *** p < 0.001; * p < 0.05; ns p > 0.05, significance determined by unpaired two-tailed t test. Numerical data in corresponding tab. ACE2, angiotensin-converting enzyme 2; AT1, alveolar type 1; AT2, alveolar type 2; PDPN, Podoplanin; PFU, plaque forming assay; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.

Article Snippet: Primary antibodies used for immunoblotting: pan-ACE2 (1:1,000; R&D Systems; AF933), hACE2 (1:1,000; Atlas Antibodies; AMAB91259), β-Actin (1:1,000; Cell Signaling; 4970).

Techniques: Immunodetection, Infection, Immunohistochemistry, Virus, Staining, Two Tailed Test

Journal: PLOS Biology

Article Title: Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection

doi: 10.1371/journal.pbio.3001989

Figure Lengend Snippet: (A, B) Weight loss and survival of hACE2 fl/y and Shh Cre/+ ; hACE2 fl/y mice after infection with 10 4 PFU of SARS-CoV-2. N = 11 ( hACE2 fl/y ) and 4 (Shh Cre/+ ; hACE2 fl/y ), two independent experiments. Note : Data for hACE2 fl/y same as Figs and S2 since Shh Cre/+ ; hACE2 fl/y animals were littermates. ( C) Pulse oximetry measured in WT, hACE2 fl/y , and Shh Cre/+ ; hACE2 fl/y mice 6 days after exposure to 10 4 PFU of SARS-CoV-2 virus. ( D) HE staining of WT, hACE2 fl/y , and Shh Cre/+ ; hACE2 fl/y lung tissue 6 days after exposure to 10 4 PFU of SARS-CoV-2 virus. Boxed regions at higher magnification in images below. Arrows, sites of focal consolidation. Asterisks, intravascular thrombi. Hashtag, acute emphysematous changes. Representative of N = 4 animals per genotype. ( E, F) Immunohistochemistry of WT, hACE2 fl/y , and Shh Cre/+ ; hACE2 fl/y lung tissue 6 days after exposure to 10 4 PFU of SARS-CoV-2 virus using antibodies against ICAM-1 and PDPN, or vWF and Endomucin (endothelial cells). Arrowheads in F identify vWF-positive microvasculature of the lung in hACE2 fl/y and Shh Cre/+ ; hACE2 fl/y animals. Representative of N = 4 animals per genotype. Scale bars in all images, 50 μm. Note: Images in each panel were taken at lower or higher magnification from the same tissue section respective to genotype. * p < 0.05, ** p < 0.001; **** p < 0.0001 by unpaired two-tailed t test, one-way ANOVA with Holm–Sidak correction for multiple comparisons, or log-rank Mantel Cox test. Numerical data in corresponding tab. HE, hematoxylin–eosin; ICAM-1, intracellular adhesion marker 1; PDPN, Podoplanin; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; vWF, von Willebrand’s Factor; WT, wild-type.

Article Snippet: Primary antibodies used for immunoblotting: pan-ACE2 (1:1,000; R&D Systems; AF933), hACE2 (1:1,000; Atlas Antibodies; AMAB91259), β-Actin (1:1,000; Cell Signaling; 4970).

Techniques: Infection, Virus, Staining, Immunohistochemistry, Two Tailed Test, Marker

Journal: PLOS Biology

Article Title: Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection

doi: 10.1371/journal.pbio.3001989

Figure Lengend Snippet: ( A, B) Immunohistochemistry of SARS-CoV-2 nucleocapsid and epithelial cell E-cadherin in the RE, OE, and OB 2 and 5–6 days after infection of hACE2 fl/y and Shh Cre/+ ; hACE2 fl/y mice. Arrowheads indicate sites of viral nucleocapsid detection. Representative of N = 4 animals per genotype and time point. Scale bars 100 μm. ( C, D) Immunohistochemistry of SARS-CoV-2 nucleocapsid, neuronal NeuN, and glial cell GFAP in the cerebral cortex (Co) 2 and 5–6 days after infection. Arrowheads indicate sites of GFAP+ reactive gliosis. Arrows indicate nucleocapsid colocalization with NeuN staining. Representative of N = 4 animals per genotype and time point. Scale bars 100 μm top, 50 μm bottom. (E) Diagram of the mouse nasal cavity and cranial anatomy. ( F) In situ hybridization detection of SARS-CoV-2 mRNA 5 days postinfection reveals virus in the OB and cerebral cortex of the brain, but not the OE of the nose. Scale bar 250 μm. Note: Images in each panel were taken at lower or higher magnification from the same tissue section respective to genotype and highlight different anatomical regions. OB, olfactory bulb of the brain; OE, olfactory epithelium; RE, respiratory epithelium; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.

Article Snippet: Primary antibodies used for immunoblotting: pan-ACE2 (1:1,000; R&D Systems; AF933), hACE2 (1:1,000; Atlas Antibodies; AMAB91259), β-Actin (1:1,000; Cell Signaling; 4970).

Techniques: Immunohistochemistry, Infection, Staining, In Situ Hybridization, Virus

Journal: PLOS Biology

Article Title: Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection

doi: 10.1371/journal.pbio.3001989

Figure Lengend Snippet: ( A) HE staining of the indicated mouse tissues was performed 24 hours after intraperitoneal injection of 100 mg/kg MMZ or vehicle control. Scale bars 100 μm for the OE, OB, and 50 μm for the lung. Representative of N = 3 per condition. (B, C) Weight loss and survival of hACE2 fl/y mice treated with MMZ or vehicle prior to infection with 10 5 PFU of SARS-CoV-2 virus. Asterisks indicate significant differences in weight between vehicle and MMZ treated hACE2 fl/y animals. N = 14 (Vehicle) and 15 (MMZ). ( D, E) Immunohistochemistry of WT, vehicle-treated hACE2 fl/y , and MMZ-treated hACE2 fl/y mouse cerebral cortex and lung 5–6 days after infection with 10 5 PFU of SARS-CoV-2 virus using antibodies that recognize viral nucleocapsid, the neuronal marker NeuN, the glial cell marker GFAP, the alveolar type II cell marker DC-LAMP, the AT1 cell marker PDPN, and the nuclear stain DAPI. Arrows indicate nucleocapsid staining colocalized with NeuN+ neurons (brain) or PDPN+ AT1 cells (lung). Representative of N = 4 per condition. ( F, G) Immunohistochemistry of WT and MMZ-treated hACE2 fl/y mouse cerebral cortex and lung 14 days after infection with 10 5 PFU of SARS-CoV-2 virus was performed as described in D and E. Note: Day 5 hACE2 fl/y samples (middle panel) were included on the same tissue slide as a positive control. Arrows indicate nucleocapsid staining colocalized with neurons and AT1 cells in vehicle-treated hACE2 fl/y mice. Representative of N = 4 per condition. Scale bars D-G 50 μm. ( H) The Foxg1 Cre/+ allele drives Cre expression in the OE and neurons of the brain (shown in red). ( I, J) Weight loss and survival of hACE2 fl/y and Foxg1 Cre/+ ; hACE2 fl/y mice after infection with 10 5 viral titer per mouse. n = 4 and 3 mice, respectively. ( K) Pulse oximetry measured in WT, hACE2 fl/y , and Foxg1 Cre/+ ; hACE2 fl/y mice 5–6 and 12 days after exposure to SARS-CoV-2 virus, respectively. * p < 0.05, ** p < 0.001; **** p < 0.0001 by unpaired two-tailed t test, one-way ANOVA with Holm–Sidak correction for multiple comparisons, or log-rank Mantel Cox test. Numerical data in corresponding tab. hACE2, human ACE2; HE, hematoxylin–eosin; MMZ, methimazole; OB, olfactory bulb; OE, olfactory epithelium; PDPN, Podoplanin; PFU, plaque-forming unit; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WT, wild-type.

Article Snippet: Primary antibodies used for immunoblotting: pan-ACE2 (1:1,000; R&D Systems; AF933), hACE2 (1:1,000; Atlas Antibodies; AMAB91259), β-Actin (1:1,000; Cell Signaling; 4970).

Techniques: Staining, Injection, Control, Infection, Virus, Immunohistochemistry, Marker, Positive Control, Expressing, Two Tailed Test

Journal: PLOS Biology

Article Title: Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection

doi: 10.1371/journal.pbio.3001989

Figure Lengend Snippet: (A) Generation of LSL-hACE2 mice using gene targeting of the mouse Rosa26 locus. WPRE, SV40, PGK-NeoR. ( B) Immunoblotting of whole lung lysates from WT, LSL-hACE2 +/0 , and Shh Cre/+ ;LSL-hACE2 +/0 mice was performed using anti-hACE2 and anti-β-actin antibodies. Each lane represents a single animal. Representative of n = 3 per genotype and two independent experiments. ( C) Immunohistochemistry of lung from LSL-hACE2 +/0 and Shh Cre/+ ;LSL-hACE2 +/0 mice was performed using anti-panACE2 and anti-hACE2 antibodies. Representative of N = 3 per genotype and two independent experiments. ( D) Immunohistochemistry of lung from LSL-hACE2 +/0 and Shh Cre/+ ;LSL-hACE2 +/0 mice was performed using antibodies to detect SARS-CoV-2 nucleocapsid, AT1 cell PDPN, and AT2 cell DC-LAMP 2 days after infection with 10 4 PFU of SARS-CoV-2 virus. Representative of n = 4 per genotype. ( E) Immunohistochemistry of lung from LSL-hACE2 +/0 and Shh Cre/+ ;LSL-hACE2 +/0 mice was performed using antibodies to detect SARS-CoV-2 nucleocapsid, AT1 cell PDPN, and AT2 cell DC-LAMP 6 days after infection with 10 4 PFU of SARS-CoV-2 virus. Representative of n = 4 per genotype. ( F) Immunohistochemistry of cerebral cortex from LSL-hACE2 +/0 and Shh Cre/+ ;LSL-hACE2 +/0 mice was performed using antibodies to detect SARS-CoV-2 nucleocapsid, neuronal NeuN, and glial cell GFAP 6 days after infection with 10 4 PFU of SARS-CoV-2 virus. Representative of n = 4 per genotype. ( G) HE staining of LSL-hACE2 +/0 and Shh Cre/+ ;LSL-hACE2 +/0 lung tissue 2 days after exposure to 10 4 PFU of SARS-CoV-2 virus. Arrows, sites of inflammatory cell infiltrate. Hashtag, acute emphysematous changes. Representative of n = 4 animals per genotype. ( H) HE staining of LSL-hACE2 +/0 and Shh Cre/+ ;LSL-hACE2 +/0 lung tissue 6 days after exposure to 10 4 PFU of SARS-CoV-2 virus. Arrows, sites of alveolar inflammatory infiltrate and hyalinosis. Representative of n = 4 animals per genotype. ( I) Pulse oximetry measured in LSL-hACE2 +/0 and Shh Cre/+ ;LSL-hACE2 +/0 mice 6, 10, and 14 days after exposure to 10 5 PFU of SARS-CoV-2 virus. ( J, K) Weight loss and survival of LSL-hACE2 +/0 and Shh Cre/+ ;LSL-hACE2 +/0 mice after infection with 10 5 PFU of SARS-CoV-2. N = 6 (LSL-hACE2 +/0 ) and 7 (Shh Cre/+ ;LSL-hACE2 +/0 mice), two independent experiments. Scale bars in all images 50 μm. Note: Images in each panel were taken at lower and/or higher magnification from the same tissue section respective to genotype and highlight different pathology. ns, not significant, p > 0.05 **, p < 0.01 by unpaired two-tailed t test, one-way ANOVA with Holm–Sidak correction for multiple comparisons, or log-rank Mantel Cox test. Numerical data in corresponding tab. AT1, alveolar type 1; AT2, alveolar type 2; hACE2, human ACE2; HE, hematoxylin–eosin; PDPN, Podoplanin; PFU, plaque-forming unit; PGK-NeoR, Phosphoglycerate kinase promoter-driven Neomycin resistance cassette; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; SV40, simian virus 40; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; WT, wild-type.

Article Snippet: Primary antibodies used for immunoblotting: pan-ACE2 (1:1,000; R&D Systems; AF933), hACE2 (1:1,000; Atlas Antibodies; AMAB91259), β-Actin (1:1,000; Cell Signaling; 4970).

Techniques: Western Blot, Immunohistochemistry, Infection, Virus, Staining, Two Tailed Test

Journal: PLOS Biology

Article Title: Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection

doi: 10.1371/journal.pbio.3001989

Figure Lengend Snippet: ( A, B) Weight loss and survival of hACE2 fl/y and Baf53b-Cre; hACE2 fl/y mice after infection with 10 4 PFU of SARS-CoV-2. N = 10 ( hACE2 fl/y ) and 12 (Baf53b-Cre; hACE2 fl/y ), three independent experiments. ( C) Pulse oximetry measured in WT, hACE2 fl/y , and Baf53b-Cre; hACE2 fl/y mice 6 days after exposure to 10 4 PFU of SARS-CoV-2 virus. ( D) Immunoblotting of whole brain lysates from hACE2 fl/y and Baf53b-Cre; hACE2 fl/y and WT mice was performed using anti-panACE2, anti-hACE2, and anti-β-actin antibodies. Each lane represents a single animal. n = 4, two independent experiments. (E) Immunohistochemistry of SARS-CoV-2 nucleocapsid, epithelial cell Krt8, and OSN OMP in the OE of WT, hACE2 fl/y , and Baf53b-Cre; hACE2 fl/y mice 6 DPI. Representative of n = 4 animals per genotype. ( F) Immunohistochemistry of SARS-CoV-2 nucleocapsid, AT1 cell PDPN, and AT2 cell DC-LAMP in the lungs of WT, hACE2 fl/y , and Baf53b-Cre; hACE2 fl/y mice 6 DPI. ( G) Immunohistochemistry of SARS-CoV-2 nucleocapsid, neuronal NeuN, and glial cell GFAP in the cerebral cortex at low and high magnification of the same tissue section of WT, hACE2 fl/y , and Baf53b-Cre; hACE2 fl/y mice 6 DPI. Representative of N = 5–6 animals per genotype and time point. Scale bars in all images 50 μm. **** p < 0.0001 by unpaired two-tailed t test, one-way ANOVA with Holm–Sidak correction for multiple comparisons, or log-rank Mantel Cox test. Numerical data in corresponding tab. AT1, alveolar type 1; AT2, alveolar type 2; DPI, days postinfection; OE, olfactory epithelium; OMP, olfactory marker protein; OSN, olfactory sensory neuron; PDPN, Podoplanin; PFU, plaque-forming unit; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WT, wild-type.

Article Snippet: Primary antibodies used for immunoblotting: pan-ACE2 (1:1,000; R&D Systems; AF933), hACE2 (1:1,000; Atlas Antibodies; AMAB91259), β-Actin (1:1,000; Cell Signaling; 4970).

Techniques: Infection, Virus, Western Blot, Immunohistochemistry, Two Tailed Test, Marker

Journal: PLOS Biology

Article Title: Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection

doi: 10.1371/journal.pbio.3001989

Figure Lengend Snippet: ( A-C) Immunohistochemistry of WT and Foxg1 Cre/+ ;LSL-hACE2 +/0 mouse OE (A), OB, and cerebral cortex (B) and lung (C) 6 days after infection with 10 5 PFU of SARS-CoV-2 using antibodies that recognize viral nucleocapsid, sustentacular cell E-cadherin, olfactory sensory neuron βIII-tubulin, neuronal NeuN, glial GFAP, AT1 PDPN, or AT2 DC-LAMP. Asterisk indicates OB with nucleocapsid staining. Arrows indicate nucleocapsid staining colocalized with βIII-tubulin+ OSNs (OE in A) or NeuN+ neurons (OB and cerebral cortex in B). White dotted lines trace the border between the OE and OB. Representative of n = 5 per genotype. ( D) HE staining of WT and Foxg1 Cre/+ ; hACE2 fl/y lung tissue 6 days after exposure to 10 5 PFU of SARS-CoV-2 virus. Arrows, sites of focal consolidation. Asterisks, intravascular thrombi. Representative of n = 5 animals per genotype. ( E, F) Immunohistochemistry of WT and Foxg1 Cre/+ ; hACE2 fl/y lung tissue 6 days after exposure to 10 5 PFU of SARS-CoV-2 virus using antibodies against ICAM-1 and PDPN, or vWF and PECAM. Arrows in I identify vWF-positive microvasculature of the lung in Foxg1 Cre/+ ; hACE2 fl/y animals. Representative of n = 5 animals per genotype. ( G, H ) Weight loss and survival of LSL-hACE2 +/0 and Foxg1 Cre/+ ; LSL-hACE2 +/0 mice after infection with 10 5 or 10 4 PFU of SARS-CoV-2. N = 4 (LSL-hACE2 +/0 ), 8 (Foxg1 Cre/+ ; LSL-hACE2 +/0 10 5 PFU), 5 (Foxg1 Cre/+ ; LSL-hACE2 +/0 10 4 PFU) and two independent experiments. Asterisks indicate time points at which significant differences in weight (C) or survival (D) were observed between infected Foxg1 Cre/+ ; LSL-hACE2 +/0 and LSL-hACE2 +/0 mice animals. ( I) Pulse oximetry of WT (LSL-hACE2 +/0 or Foxg1 Cre/+ ) and Foxg1 Cre/+ ; LSL-hACE2 +/0 mice infected with 10 4 viral titer at the time of harvest (Day 6–8). *** p < 0.001; **** p < 0.0001 determined by unpaired, two-tailed t test or log-rank Mantel Cox test. Scale bars in all images 50 μm. Numerical data in corresponding tab. hACE2, human ACE2; ICAM-1, intracellular adhesion marker 1; OB, olfactory bulb; OE, olfactory epithelium; OSN, olfactory sensory neuron; PDPN, Podoplanin; PECAM, platelet endothelial cell adhesion molecule; PFU, plaque-forming unit; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; vWF, von Willebrand’s Factor; WT, wild-type.

Article Snippet: Primary antibodies used for immunoblotting: pan-ACE2 (1:1,000; R&D Systems; AF933), hACE2 (1:1,000; Atlas Antibodies; AMAB91259), β-Actin (1:1,000; Cell Signaling; 4970).

Techniques: Immunohistochemistry, Infection, Staining, Virus, Two Tailed Test, Marker

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Generation of the SARS-CoV-2 N conditional knock-in mouse model. a Schematic diagrams illustrating the knock-in strategy, in which the CAG-loxP-Stop-loxP-SARS-CoV-2 N-WPRE-PolyA sequence was inserted into the Tigre locus on chromosome 9, using the K18-hACE2 KI mouse background (N-hACE2 mouse). The stop sequence was flanked by two loxP sites, and upon expression of Cre recombinase, the stop sequence located between these loxP sites was excised. N-hACE2 mice were crossed with Rosa26 SA-CreERT2 or Sftpc-IRES-iCre mice, resulting in the generation of SA-N-hACE2 (TAM-inducible systemic Cre expression) and Sftpc-N-hACE2 (lung-specific Cre expression) mice. b N-hACE2 mouse genotyping by PCR showed the presence of N gene and hACE2 gene fragments. M: GimiRun DM5000 DNA Marker. c PCR confirmed excision of the stop sequence between loxP sites in SA-N-hACE2 mice after 5 days of TAM injection. Western blot analysis of SARS-CoV-2 N protein expression in multiple tissues from SA-N-hACE2 mice after TAM induction ( d ) and from Sftpc-N-hACE2 mice ( e ). f Immunofluorescence staining of lung sections was conducted using 4,6-diamidino-2-phenylindole (DAPI, blue), an anti-SARS-CoV-2 N antibody (red), and an anti-hACE2 antibody (green) to evaluate the expression of SARS-CoV-2 N and hACE2 in lung cells

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Knock-In, Sequencing, Expressing, Marker, Injection, Western Blot, Immunofluorescence, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: SA-N-hACE2 mice are susceptible to SARS-CoV-2 ΔN/GFP-HiBiT infection. a Experimental design for intranasal infection. After TAM treatment, SA-N-hACE2 mice were infected either with 5 × 10 4 or 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. Tissue samples were collected at the indicated dpi. b Changes in the weights of the mice are shown ( n = 4 per group). c qRT‒PCR was used to quantify viral loads in tissues at 7 dpi ( n = 4 per group). The viral loads ( d ) and luminescence ( e ) were measured in the lungs collected at 2, 4, and 7 dpi ( n = 4 per group). f Immunofluorescence staining of lung sections at 2, 4, and 7 dpi was conducted using DAPI (blue) and an anti-GFP antibody (green) specific to GFP in SARS-CoV-2 ΔN/GFP-HiBiT. g – j Pathological changes observed using H&E staining in lung ( g ) and brain ( i ) tissues from SA-N-hACE2 mice challenged with 1 × 10 6 TCID 50 at 0, 2, 4, and 7 dpi. Pathology scores for the lungs ( h ) and brain ( j ) were calculated ( n = 4 per group). Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( h ). ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ND not detected

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Immunofluorescence, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Sftpc-N-hACE2 mice are susceptible to SARS-CoV-2 ΔN/GFP-HiBiT infection. a Illustration of the protocol for intranasal infection with tissue samples collected at the indicated dpi. Sftpc-N-hACE2 mice were infected with 5 × 10 4 or 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. b Changes in the body weights of the mice are shown ( n = 4 per group). c Viral loads in the tissues obtained at 7 dpi were quantified via qRT‒PCR ( n = 4 per group). The viral loads ( d ) and luminescence ( e ) were measured in the lungs collected at 2, 4, and 7 dpi ( n = 4 per group). f Immunofluorescence staining of lung sections at 2, 4, and 7 dpi was conducted using DAPI (blue) and an anti-GFP antibody (green) specific to GFP in SARS-CoV-2 ΔN/GFP-HiBiT. g – j Pathological changes in the lungs ( g ) and brains ( i ) of Sftpc-N-hACE2 mice challenged with 1 × 10 6 TCID 50 at 0, 2, 4, and 7 dpi were assessed using H&E staining. Pathology scores for the lungs ( h ) and brains ( j ) are shown ( n = 4 per group). Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( h ). ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ND not detected

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Immunofluorescence, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: SARS-CoV-2 ΔN/GFP-HiBiT infection in Sftpc-N-hACE2 mice can lead to lethal disease. a The schematic outlines the procedure for infecting the mice with 5 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. At 0, 7, 10, 14, and 21 dpi, four mice were euthanized at each time point for sample collection. Mice that experienced a loss of more than 20% of their initial body weight were euthanized as a humane endpoint. Mice were monitored for body weight changes ( b ) and survival ( c ) ( n = 16). E gene copies in lung ( d ) and brain ( e ) tissues were quantified via qRT‒PCR. f – i Pathological changes were assessed using H&E staining in the lungs ( f ) and brains ( g ). Pathology scores for the lungs ( h ) and brains ( i ) were recorded ( n = 4 per group). j Immunofluorescence staining of brain sections was performed using DAPI (blue) and an anti-GFP antibody (green) specific to GFP in SARS-CoV-2 ΔN/GFP-HiBiT. k Immunofluorescence analysis was performed on brain sections from mock-infected mice, K18-hACE2 KI mice challenged with live SARS-CoV-2, and both SA-N-hACE2 and Sftpc-N-hACE2 mice exposed to SARS-CoV-2 ΔN/GFP-HiBiT. The sections were stained with anti-IBA1 (red) and anti-CD68 (green) antibodies. l Light-sheet imaging of cleared lung tissues from Sftpc-N-hACE2 mice at 7 dpi confirmed the distribution of SARS-CoV-2 N in lung epithelial cells and SARS-CoV-2 ΔN/GFP-HiBiT infection. Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( h ). ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ND not detected

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Staining, Immunofluorescence, Imaging

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Transcriptomic profiling of the lungs of SA-N-hACE2 and Sftpc-N-hACE2 mice revealed distinct molecular features. a Scheme illustrating the transcriptome sequencing. Gene expression heatmaps of IFN-I-related genes ( b ) and cytokines and chemokines ( c ) in the lungs of the mice. Mock: PBS-treated mice; the SA-N-hACE2 and Sftpc-N-hACE2 mice were infected with 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. d Bubble plot showing gene set enrichment analysis (GSEA) results for pathways enriched in the lungs of the infected mice. The color scale indicates normalized enrichment scores (NES), while the size of the bubbles corresponds to −log 10 ( p values)

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Sequencing, Gene Expression, Infection

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Assessment of anti-SARS-CoV-2 therapeutics using SA-N-hACE2 and Sftpc-N-hACE2 mouse models. a This schematic diagram illustrates the procedure for evaluating the neutralizing antibody 7B3 and NMV. SA-N-hACE2, Sftpc-N-hACE2, and K18-hACE2 KI mice received 10 mg/kg of 7B3 at 1 dpi or 300 mg/kg NMV via oral gavage from 0–3 dpi. SA-N-hACE2 and Sftpc-N-hACE2 mice were challenged with 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT, whereas K18-hACE2 KI mice were infected with 3.57 × 10 2 TCID 50 of SARS-CoV-2 WT at 0 dpi. At 4 dpi, all mice were sacrificed, and lung samples were collected. The expression of the viral E gene in lung tissues from mock-, NMV- and 7B3-treated SA-N-hACE2 ( b ), Sftpc-N-hACE2 ( c ) and K18-hACE2 KI mice ( d ) was quantified via qRT‒PCR ( n = 4 per group). e – j Lung tissues from mock-, NMV-, and 7B3-treated SA-N-hACE2 ( e ), Sftpc-N-hACE2 ( f ), and K18-hACE2 KI ( g ) mice were analyzed using H&E staining to assess pathological changes, with histological scores documented for each group ( n = 4 per group) ( h – j ). Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( b – d , h – j ). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Expressing, Staining

Journal: Med (New York, N.y.)

Article Title: A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice

doi: 10.1016/j.medj.2020.12.006

Figure Lengend Snippet:

Article Snippet: Human Angiotensin-converting enzyme 2 (ACE2) expression plasmid , Addgene , Plasmid#1786; RRID:Addgene_1786.

Techniques: Blocking Assay, Antibody Labeling, Activation Assay, Marker, Control, Purification, Conjugation Assay, Virus, Recombinant, Staining, Transfection, Vaccines, Luciferase, Gene Expression, Expressing, Plasmid Preparation, Software