Journal: PLoS ONE

Article Title: Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

doi: 10.1371/journal.pone.0073706

Figure Lengend Snippet: ( A ) RAW264.7 cells were cultured for 24 h in the absence (V, vehicle alone) and presence of each drug (10 µM). The cells were lysed, and levels of CD9 and CD81 were examined by immunoblotting. Blots of results with fluvastatin (Fluv) and simvastatin (Simv) are shown. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) After testing 1,165 drugs, levels of CD9 and CD81 relative to actin were quantified by densitometry. Fold changes of the expression levels compared with vehicle alone were calculated and plotted. Drugs that increased the level of either CD9 or CD81 more than 1.5-fold compared with vehicle alone were regarded as positive. Correlation between fold changes in CD9 and CD81 levels was analyzed using Pearson’s correlation coefficient. ( C ) RAW264.7 cells were cultured in the absence (V) or presence of multiple statins (10 µM) and levels of CD9 and CD81 were examined by immunoblotting. The statins are arranged in order of decreasing lipophilicity. Ceri, cerivastatin; Simv, simvastatin; Fluv, fluvastatin; Ator, atorvastatin; Rosu, rosuvastatin; Prav, pravastatin. ( D ) RAW264.7 cells were cultured in the absence (shaded histograms) or presence (10 µM) of fluvastatin (open red histograms) and simvastatin (open blue histograms). Surface levels of CD9, CD63, CD81, and the integrin β1 subunit were analyzed by flow cytometry.

Article Snippet: The primary Abs were rat anti-mouse CD9 mAb (KMC8; BD Biosciences), hamster anti-mouse CD81 mAb (Eat2; UK-Serotec), rabbit anti-LAMP3 (CD63) polyclonal Ab (12632-1-AP; Proteintech), mouse anti-human CD9 mAb (MM2/57; Biosource), mouse anti-human CD81 mAb (JS64; Immunotech), rabbit anti-IκBα polyclonal Ab (9242; Cell Signaling Technology), rat anti-mouse integrin β2 subunit mAb (M18/2; BD Biosciences), rat anti-CD14 mAb (rmC5-3; BD Biosciences), rabbit anti-TLR4 polyclonal Ab (IMG577; Imgenex), mouse anti-flotillin-1 mAb (clone 18; BD Biosciences), and goat anti-CD45 polyclonal Ab (R&D Systems).

Techniques: Cell Culture, Western Blot, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

doi: 10.1371/journal.pone.0073706

Figure Lengend Snippet: ( A ) RAW264.7 cells were cultured for 24 h in the absence or presence of increasing concentrations of fluvastatin (Fluv) or simvastatin (Simv). The cells were lysed, and levels of CD9, CD63, and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) RAW264.7 cells were untreated (-) or cultured in the absence or presence of increasing concentrations of fluvastatin or simvastatin and stimulated for 24 h with 0.1 µg/ml LPS (+). Levels of CD9, CD63, and CD81 were examined by immunoblotting. Note that LPS downregulates CD9 and CD81 in the absence of statins (arrowheads). ( C ) RAW264.7 cells were cultured in the absence (-) or presence of 3 µM fluvastatin (+), and unstimulated (-) or stimulated for 24 h with 1 µg/ml LPS (+). mRNA levels of CD9 and CD81 were examined by reverse transcription PCR. GAPDH is an internal loading control. ( D ) RAW264.7 cells were cultured in the absence or presence of fluvastatin, and unstimulated or stimulated with LPS. Control (Cont) was an untreated culture. mRNA levels of CD9 and CD81 were examined by real-time PCR. Data shown are from one representative of three similar experiments. ( E ) Human monocytic THP-1 cells were treated for 4 h with 1 µg/ml phorbol 12-myristate 13-acetate, allowed to attach to a plate, and then cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting. ( F ) Mouse 3T3 fibroblasts were cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting.

Article Snippet: The primary Abs were rat anti-mouse CD9 mAb (KMC8; BD Biosciences), hamster anti-mouse CD81 mAb (Eat2; UK-Serotec), rabbit anti-LAMP3 (CD63) polyclonal Ab (12632-1-AP; Proteintech), mouse anti-human CD9 mAb (MM2/57; Biosource), mouse anti-human CD81 mAb (JS64; Immunotech), rabbit anti-IκBα polyclonal Ab (9242; Cell Signaling Technology), rat anti-mouse integrin β2 subunit mAb (M18/2; BD Biosciences), rat anti-CD14 mAb (rmC5-3; BD Biosciences), rabbit anti-TLR4 polyclonal Ab (IMG577; Imgenex), mouse anti-flotillin-1 mAb (clone 18; BD Biosciences), and goat anti-CD45 polyclonal Ab (R&D Systems).

Techniques: Cell Culture, Western Blot, Reverse Transcription, Control, Real-time Polymerase Chain Reaction

Journal: Cell

Article Title: Single-Cell Analysis of Crohn’s Disease Lesions Identifies a Pathogenic Cellular Module Associated with Resistance to Anti-TNF Therapy

doi: 10.1016/j.cell.2019.08.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies were: anti-human CD3 (clone 2GV6, Ventana), anti-human CD20 (clone L26, Dako), anti-human DC-LAMP (clone 1010E1.01, Novus biologicals), anti-human CD68 (clone KP1, Dako), anti-human HLA-DR (clone TAL1B5, Abcam), anti-Ki67 (clone 30–09, Ventana), anti-human CD206 (polyclonal, Abcam) anti-PDPN (clone D2–40, Ventana).

Techniques: Recombinant, Multiplex Assay, Software

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: LAMP3-positive material is released in minor salivary glands from SjD patients. A representative immunofluorescent image of minor salivary glands from SjD patients ( n = 11) stained with anti-LAMP3 antibody (scale bar: 50 μm). The image shows accumulation of LAMP3 in the lumen of the gland (the arrows) and in the epithelial cells (the arrowheads).

Article Snippet: Recombinant LAMP3 (recombinant DC-LAMP, rLAMP3), recombinant LAMP1 (rLAMP1) and recombinant EGFP (rEGFP) were purchased from Novus Biological (Centennial, CO, USA).

Techniques: Staining

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: Culture medium of LAMP3-overexpressing A253 cells induce apoptosis in naïve A253 cells. A253 cells were transfected with control empty plasmid, LAMP3 -encoding plasmid or LAMP1 -encoding plasmid. ( A ) Representative Western blots of transfected A253 cells (uncropped images are provided in Supplementary Fig. ) and schematic methods of the following in vitro assays. ( B ) Naïve A253 cells were treated with culture medium of control or LAMP3-overexpressing A253 cells for 48 h ( n = 3). ( C ) Naïve A253 cells were treated with culture medium of control, LAMP1-overexpressing, or LAMP3-overexpressing A253 cells or with post-ultracentrifugation supernatant or post-ultracentrifugation pellet from LAMP3-overexpressing A253 cells for 48 h ( n = 4). Apoptotic cells in naïve cell culture were determined by flow cytometry using APC Annexin V/7-AAD. Graphs show difference in mean (± SD) number of Annexin V + /7-AAD + cells in cell culture compared with control. ** p < 0.01 (one-way ANOVA).

Article Snippet: Recombinant LAMP3 (recombinant DC-LAMP, rLAMP3), recombinant LAMP1 (rLAMP1) and recombinant EGFP (rEGFP) were purchased from Novus Biological (Centennial, CO, USA).

Techniques: Transfection, Control, Plasmid Preparation, Western Blot, In Vitro, Cell Culture, Flow Cytometry

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: A fraction of extracellular particles from LAMP3-overexpressing A253 cells induces apoptosis in naïve A253 cells. ( A ) Schematic methods. Two fractions of extracellular particles (EPs) were separated from culture medium of control, LAMP3-overexpressing, or LAMP1-overexpressing A253 cells using centrifugation in combination with or without total exosome isolation reagent. Naïve A253 cells were treated with ( B ) EP fraction 2, ( C ) EP fraction 1 or ( D ) heat-denatured (heat-treated) EP fraction 1. Apoptotic cells in naïve cell culture were determined by flow cytometry using APC Annexin V/7-AAD 72 h after incubation. Graphs show difference in mean (± SD) number of Annexin V + /7-AAD + cells in cell culture compared with control ( n = 3 for all the experiments). ** p < 0.01 (one-way ANOVA).

Article Snippet: Recombinant LAMP3 (recombinant DC-LAMP, rLAMP3), recombinant LAMP1 (rLAMP1) and recombinant EGFP (rEGFP) were purchased from Novus Biological (Centennial, CO, USA).

Techniques: Control, Centrifugation, Isolation, Cell Culture, Flow Cytometry, Incubation

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: Extracellular particles from LAMP3-overexpressing cells contain LAMP3. ( A ) Western blot analysis of EPs separated from the culture medium of control, LAMP3-overexpressing, and LAMP1-overexpressing A253 cells using centrifugation in combination with or without total exosome isolation reagent. Equal volume of protein loaded in each lane. Lower panel shows same membrane as the one used in Western blot analysis but stained with Reversible Protein Stain Kit. Uncropped images are provided in Supplementary Fig. . ( B ) Cell surface LAMP3 expression was analyzed by flow cytometry (bold line: LAMP3 staining, filled area with dashed line: isotype control) and under an immunofluorescent microscopy (scale bar: 100 μm) in control, LAMP1-overexpressing, and LAMP3-overexpressing A253 cells 48 h post-transfection. ( C ) EPs derived from control and LAMP3-overexpressing A253 cells were immunoprecipitated using anti-LAMP3 monoclonal antibody-conjugated beads. Presence of LAMP3 protein on EP membrane surface was determined by analyzing beads stained with anti-LAMP3 polyclonal antibody using flow cytometry. Graph showing difference in mean (± SD) percentage of LAMP3-positive beads compared with beads bound to isotype control ( n = 4). ** p < 0.01 (one-way ANOVA).

Article Snippet: Recombinant LAMP3 (recombinant DC-LAMP, rLAMP3), recombinant LAMP1 (rLAMP1) and recombinant EGFP (rEGFP) were purchased from Novus Biological (Centennial, CO, USA).

Techniques: Western Blot, Control, Centrifugation, Isolation, Membrane, Staining, Expressing, Flow Cytometry, Microscopy, Transfection, Derivative Assay, Immunoprecipitation

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: Extracellular particles from LAMP3-overexpressing transfer LAMP3 to naïve cells. ( A ) Western blot analysis of naïve A253 cells treated with EPs from control or LAMP3-overexpressing A253 cells. “※” indicates non-specific bands. ( B ) Western blot analysis of LAMP3-GFP expression in A253 cells transfected with empty plasmid (control) or pLenti-LAMP3-mGFP plasmid (A253-LAMP3-mGFP cells). ( C ) Western blot analysis of EPs derived from A253-LAMP3-mGFP cells. ( D ) A253-LAMP3-mGFP cells were imaged in real time to visualize EP release. A particle containing LAMP3-mGFP is seen being released from the cell into the extracellular environment (scale bar: 5 μm). ( E ) Naïve A253 cells were treated with LAMP3-mGFP–containing particle and imaged in real time to visualize particle uptake. Uptake of a LAMP3-mGFP–containing particle by a naïve cell and subsequent membrane blebbing are shown (scale bar: 10 μm). Uncropped images of Western blots are provided in Supplementary Fig. .

Article Snippet: Recombinant LAMP3 (recombinant DC-LAMP, rLAMP3), recombinant LAMP1 (rLAMP1) and recombinant EGFP (rEGFP) were purchased from Novus Biological (Centennial, CO, USA).

Techniques: Western Blot, Control, Expressing, Transfection, Plasmid Preparation, Derivative Assay, Membrane

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: Transfection of recombinant LAMP3 protein induces apoptosis in A253 cells. ( A ) Representative Western blots of A253 cells transfected with recombinant LAMP3 (rLAMP3), recombinant LAMP1 (rLAMP1), or control FLAG peptide (uncropped images are provided in Supplementary Fig. ). ( B ) Apoptotic cells were determined in A253 cell culture by flow cytometry using APC Annexin V/7-AAD 14 h after transfection. Graph showing difference in mean (± SD) number of Annexin V + /7-AAD + cells in A253 cell culture transfected with rLAMP3 or rLAMP1 compared with control. ( C ) A253 cells were transfected with rLAMP3, heat-treated rLAMP3, or FLAG peptide. Apoptotic cells were determined in A253 cell culture by flow cytometry using APC Annexin V/7-AAD 14 h after transfection. Graph showing difference in mean (± SD) number of Annexin V + /7-AAD + cells in A253 cell culture transfected with rLAMP3 or heat-treated rLAMP3 compared with control. ( D ) A253 cells were treated with control FLAG peptide or rLAMP3 alone (without transfection reagent) or transfected with control FLAG peptide or rLAMP3 with transection reagent. After 14 h, number of apoptotic cells was determined by flow cytometry using APC Annexin V/7-AAD. Graph showing difference in mean (± SD) number of Annexin V + /7-AAD + cells in cell culture treated with rLAMP3 alone or transfected with FLAG peptide or rLAMP3 compared with FLAG peptide alone. ** p < 0.01 (one-way ANOVA, n = 4 for all the experiments).

Article Snippet: Recombinant LAMP3 (recombinant DC-LAMP, rLAMP3), recombinant LAMP1 (rLAMP1) and recombinant EGFP (rEGFP) were purchased from Novus Biological (Centennial, CO, USA).

Techniques: Transfection, Recombinant, Western Blot, Control, Cell Culture, Flow Cytometry

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: LAMP3 transfer via extracellular particles induces caspase-dependent apoptosis. ( A ) Naïve A253 cells were treated with EPs from control and LAMP3-overexpressing A253 cells in combination with or without zVAD-fmk (zVAD), a pan-caspase inhibitor. Apoptotic cells were determined by flow cytometry using APC Annexin V/7-AAD 72 h after incubation. Graph showing difference in mean (± SD) number of Annexin V + /7-AAD + cells in naïve A253 cell culture compared with control ( n = 5). ( B ) Naïve A253 cells were treated with EPs from control and LAMP3-overexpressing A253 cells for 6 h. Galectin-3 puncta formation in treated A253 cell culture was visualized by immunofluorescence (scale bar: 50 μm). Graph showing percentage of galectin-3 puncta–positive cells per 100 treated A253 cells ( n = 3). ** p < 0.01 (one-way ANOVA).

Article Snippet: Recombinant LAMP3 (recombinant DC-LAMP, rLAMP3), recombinant LAMP1 (rLAMP1) and recombinant EGFP (rEGFP) were purchased from Novus Biological (Centennial, CO, USA).

Techniques: Control, Flow Cytometry, Incubation, Cell Culture, Immunofluorescence

Journal: Scientific Reports

Article Title: LAMP3 transfer via extracellular particles induces apoptosis in Sjögren’s disease

doi: 10.1038/s41598-023-28857-w

Figure Lengend Snippet: Graphical summary. Extracellular particles containing LAMP3 are released by LAMP3-overexpressing cells and taken up by a neighboring LAMP3-naïve cell. This results in LAMP3 protein transfer to the cytoplasm of the naïve cell. Here, LAMP3 induces lysosomal membrane permeabilization (LMP), leading to apoptotic cell death.

Article Snippet: Recombinant LAMP3 (recombinant DC-LAMP, rLAMP3), recombinant LAMP1 (rLAMP1) and recombinant EGFP (rEGFP) were purchased from Novus Biological (Centennial, CO, USA).

Techniques: Membrane

Journal: The Journal of international medical research

Article Title: Expression of dendritic cell lysosome-associated membrane protein and dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin in condyloma acuminatum lesions.

doi: 10.1177/0300060513476991

Figure Lengend Snippet: Figure 1. Immunohistochemical staining of dendritic cell lysosome-associated membrane protein (DC-LAMP) in (A) normal foreskin tissue and (B) condyloma acuminatum lesion; arrows show DC-LAMP in epidermis and dermis. Immunohistochemical staining of dendritic cell-specific intercellular adhesion molecule- 3 grabbing nonintegrin (DC-SIGN) protein in (C) normal foreskin tissue and (D) condyloma acuminatum lesion; arrows show DC-SIGN in epidermis and dermis.

Article Snippet: Separated proteins were then transferred onto a polyvinylidene fluoride (PVDF) membrane at 40V for 2 h. After blocking with 5% low-fat dry milk for 1 h, the PVDF membranes were incubated with at Glasgow University Library on May 20, 2015imr.sagepub.comDownloaded from a monoclonal antihuman DC-LAMP antibody (1 : 500 dilution; R&D Systems), monoclonal antihuman DC-SIGN antibody (1 : 500 dilution; R&D Systems), or mouse antihuman b-actin antibody (C4) (1 : 400 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 C. Membranes were washed with 50mM Tris,150mM NaCl, and 0.05% Tween 20, pH 7.6 (TBST) five times for 5min, then incubated with horseradish peroxidaselabelled goat antirabbit immunoglobulin (HþL) antibody (1 : 50 dilution; Beyotime) for 1 h at 37 C. Blots were washed three times for 5min in TBST and developed with DAB to visualize the protein bands.

Techniques: Immunohistochemical staining, Staining, Membrane

Journal: bioRxiv

Article Title: SARS-CoV-2 infection of olfactory epithelial cells and neurons drives acute lung injury and lethal COVID-19 in mice

doi: 10.1101/2021.12.04.471245

Figure Lengend Snippet: a , 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 (alveolar type 2 cells) and PDPN (alveolar type 1 cells) as well as the nuclear stain DAPI. N=4 for both genotypes, one experiment b , 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. c , 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 (alveolar type 2 cells) and PDPN (alveolar type 1 cells) as well as the nuclear stain DAPI. N=6 for all genotypes, two independent experiments. d , 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. e , Infectious viral load was measured by plaque forming assay (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. Scale bars, 50 µm. ***p<0.001; *p<0.05; ns p>0.05, significance determined by unpaired two-tailed t-test.

Article Snippet: Primary antibodies: pan-ACE2 (goat, 1:1000, R&D AF933), human-ACE2 (rabbit, 1:200, Abcam ab108209), E-cadherin (rabbit, 1:200, Cell Signaling 3195S), E-cadherin (goat, 1:200, R&D AF748), Olfactory marker protein (goat,1:200, Wako 544-10001), SARS-CoV-2 nucleocapsid (rabbit, 1:500, Rockland 200-401-A50), Podoplanin (hamster, 1:500, Novus Biologicals AB15858), DC-LAMP (rat, 1:25, Novus Biologicals, DDX0191P-100), ICAM-1 (rabbit, 1:500, Abcam ab179707), vWF (rabbit 1:1000, Novus Biologicals NB600-586), Endomucin (rat, 1:100, Abcam ab106100), β3-Tubulin (mouse, 1:1000, Abcam ab78078), NeuN (mouse, 1:1000, Novus Biologicals NBP1-92693), GFAP (chicken, 1:1000, Novus Biologicals NBP1-05198), GFP (goat 1:500, Rockland, 600-101-215)

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

Journal: bioRxiv

Article Title: SARS-CoV-2 infection of olfactory epithelial cells and neurons drives acute lung injury and lethal COVID-19 in mice

doi: 10.1101/2021.12.04.471245

Figure Lengend Snippet: a , H&E staining of the indicated mouse tissues was performed 24 hours after intraperitoneal injection of 100 mg/kg methimazole (MMZ) or vehicle control. Scale bars 100 µm for the olfactory epithelium, olfactory bulb 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 24 hours 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 wild-type, 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 alveolar type I (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 wild-type 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-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 olfactory epithelium (OE) and neurons of the brain (shown in red). i-j, Weight loss and survival of hACE2 fl/y and 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 wild-type (WT), hACE2 fl/y and Foxg1 Cre/+ ; hACE2 fl/y mice 5-6 days after exposure to SARS-CoV-2 virus. *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.

Article Snippet: Primary antibodies: pan-ACE2 (goat, 1:1000, R&D AF933), human-ACE2 (rabbit, 1:200, Abcam ab108209), E-cadherin (rabbit, 1:200, Cell Signaling 3195S), E-cadherin (goat, 1:200, R&D AF748), Olfactory marker protein (goat,1:200, Wako 544-10001), SARS-CoV-2 nucleocapsid (rabbit, 1:500, Rockland 200-401-A50), Podoplanin (hamster, 1:500, Novus Biologicals AB15858), DC-LAMP (rat, 1:25, Novus Biologicals, DDX0191P-100), ICAM-1 (rabbit, 1:500, Abcam ab179707), vWF (rabbit 1:1000, Novus Biologicals NB600-586), Endomucin (rat, 1:100, Abcam ab106100), β3-Tubulin (mouse, 1:1000, Abcam ab78078), NeuN (mouse, 1:1000, Novus Biologicals NBP1-92693), GFAP (chicken, 1:1000, Novus Biologicals NBP1-05198), GFP (goat 1:500, Rockland, 600-101-215)

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

Journal: bioRxiv

Article Title: SARS-CoV-2 infection of olfactory epithelial cells and neurons drives acute lung injury and lethal COVID-19 in mice

doi: 10.1101/2021.12.04.471245

Figure Lengend Snippet: a-b, Cerebral cortex (a) and lung (b) immunohistochemistry of hACE2 fl/y animals treated with vehicle or methimazole (MMZ) followed by infection with 10 5 viral titer. The tissues shown on the far right (“ hACE2 fl + MMZ 8 DPI”) are from a hACE2 fl mouse that exhibited weight loss and required euthanasia 8 days following infection with 10 5 SARS-CoV-2 virus despite pre-treatment with MMZ. Cerebral cortex stained with viral nucleocapsid, neuronal NeuN and glial cell GFAP. Lung stained with viral nucleocapsid, alveolar type I cells PDPN, and alveolar type II cell DC-LAMP. Arrows indicate nucleocapsid staining colocalized with NeuN+ neurons or PDPN+ AT1 cells. N=4 per genotype except for N=1 (MMZ treated). Scale bars 50 µm.

Article Snippet: Primary antibodies: pan-ACE2 (goat, 1:1000, R&D AF933), human-ACE2 (rabbit, 1:200, Abcam ab108209), E-cadherin (rabbit, 1:200, Cell Signaling 3195S), E-cadherin (goat, 1:200, R&D AF748), Olfactory marker protein (goat,1:200, Wako 544-10001), SARS-CoV-2 nucleocapsid (rabbit, 1:500, Rockland 200-401-A50), Podoplanin (hamster, 1:500, Novus Biologicals AB15858), DC-LAMP (rat, 1:25, Novus Biologicals, DDX0191P-100), ICAM-1 (rabbit, 1:500, Abcam ab179707), vWF (rabbit 1:1000, Novus Biologicals NB600-586), Endomucin (rat, 1:100, Abcam ab106100), β3-Tubulin (mouse, 1:1000, Abcam ab78078), NeuN (mouse, 1:1000, Novus Biologicals NBP1-92693), GFAP (chicken, 1:1000, Novus Biologicals NBP1-05198), GFP (goat 1:500, Rockland, 600-101-215)

Techniques: Immunohistochemistry, Infection, Virus, Staining

Journal: bioRxiv

Article Title: SARS-CoV-2 infection of olfactory epithelial cells and neurons drives acute lung injury and lethal COVID-19 in mice

doi: 10.1101/2021.12.04.471245

Figure Lengend Snippet: a , Generation of LSL-hACE2 mice using gene targeting of the mouse Rosa26 locus. Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), Simian virus 40 (SV40), Phosphoglycerate kinase promoter driven Neomycin resistance cassette (PGK-NeoR). b-d , Immunohistochemistry of olfactory epithelium, cerebral cortex and lung from Foxg1 Cre/+ ;LSL-hACE2 +/0 and LSL-hACE2 +/0 mouse brain tissue was performed using antibodies that recognize human ACE2, (hACE2), both human and mouse ACE2 (pan-ACE2), the neuronal marker NeuN, alveolar type 1 cell Aquaporin 5 (AQP5), and alveolar type II cells (DC-LAMP). Representative of N=2 per genotype. Scale bars 50 µm. e-f , Immunohistochemistry of Foxg1 Cre/+ ;LSL-hACE2 +/0 and LSL-hACE2 +/0 mouse olfactory epithelium, olfactory bulb, and cerebral cortex 5-6 days after infection with 10 5 PFU of SARS-CoV-2 virus using antibodies that recognize viral nucleocapsid, sustentacular cell E-cadherin, olfactory sensory neuron (OSN) βIII-tubulin, neuronal NeuN, or glial GFAP. OE-olfactory epithelium. OB-olfactory bulb. Asterisk indicates olfactory bulb with nucleocapsid staining. Arrows indicate nucleocapsid staining colocalized with βIII-tubulin+ OSNs (olfactory epithelium) or NeuN+ neurons (brain). Red dotted lines trace nucleocapsid staining colocalized within β3-tubulin+ OSN axons. Representative of N=4 per genotype. Scale bars 50 µm. 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. Asterisks indicate timepoints at which significant differences in weight were observed between infected Foxg1 Cre/+ ; LSL-hACE2 +/0 and LSL-hACE2 +/0 mice animals. *** P<0.001; **** P<0.0001 determined by unpaired, two-tailed t-test or log-rank Mantel Cox test. 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<0.0001 determined by unpaired, two-tailed t-test.

Article Snippet: Primary antibodies: pan-ACE2 (goat, 1:1000, R&D AF933), human-ACE2 (rabbit, 1:200, Abcam ab108209), E-cadherin (rabbit, 1:200, Cell Signaling 3195S), E-cadherin (goat, 1:200, R&D AF748), Olfactory marker protein (goat,1:200, Wako 544-10001), SARS-CoV-2 nucleocapsid (rabbit, 1:500, Rockland 200-401-A50), Podoplanin (hamster, 1:500, Novus Biologicals AB15858), DC-LAMP (rat, 1:25, Novus Biologicals, DDX0191P-100), ICAM-1 (rabbit, 1:500, Abcam ab179707), vWF (rabbit 1:1000, Novus Biologicals NB600-586), Endomucin (rat, 1:100, Abcam ab106100), β3-Tubulin (mouse, 1:1000, Abcam ab78078), NeuN (mouse, 1:1000, Novus Biologicals NBP1-92693), GFAP (chicken, 1:1000, Novus Biologicals NBP1-05198), GFP (goat 1:500, Rockland, 600-101-215)

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

Journal: bioRxiv

Article Title: SARS-CoV-2 infection of olfactory epithelial cells and neurons drives acute lung injury and lethal COVID-19 in mice

doi: 10.1101/2021.12.04.471245

Figure Lengend Snippet: a , Hematoxylin-eosin (H&E) staining of wild-type and Foxg1 Cre/+ ; LSL-hACE2 +/0 lung tissue 6 days after exposure to 10 5 PFU of SARS-CoV-2 virus. Arrows, sites of focal consolidation. Asterisks, intravascular thrombi. Hashtag, acute emphysematous changes. Representative of N=4 animals per genotype. Scale bars 100 µm. b, Immunohistochemistry of wild-type and Foxg1 Cre/+ ; LSL-hACE2 +/0 lung tissue 6 days after exposure to 10 5 PFU of SARS-CoV-2 virus using antibodies against viral nucleocapsid, alveolar type 1 PDPN, and alveolar type II DC-LAMP. Representative of N=4 animals per genotype, Scale bars 50 µm. c-d , Immunohistochemistry of wild-type and Foxg1 Cre/+ ; LSL-hACE2 +/0 lung tissue 6 days after exposure to 10 5 PFU of SARS-CoV-2 virus using antibodies against Intracellular adhesion marker-1 (ICAM-1) and Podoplanin (PDPN), or von Willebrand’s Factor (vWF) and PECAM (endothelial cells). Arrowheads in d identify vWF-positive microvasculature of the lung in Foxg1 Cre/+ ; LSL-hACE2 +/0 animals. Representative of N=3-4 animals per genotype. Scale bars, 50 µm.

Article Snippet: Primary antibodies: pan-ACE2 (goat, 1:1000, R&D AF933), human-ACE2 (rabbit, 1:200, Abcam ab108209), E-cadherin (rabbit, 1:200, Cell Signaling 3195S), E-cadherin (goat, 1:200, R&D AF748), Olfactory marker protein (goat,1:200, Wako 544-10001), SARS-CoV-2 nucleocapsid (rabbit, 1:500, Rockland 200-401-A50), Podoplanin (hamster, 1:500, Novus Biologicals AB15858), DC-LAMP (rat, 1:25, Novus Biologicals, DDX0191P-100), ICAM-1 (rabbit, 1:500, Abcam ab179707), vWF (rabbit 1:1000, Novus Biologicals NB600-586), Endomucin (rat, 1:100, Abcam ab106100), β3-Tubulin (mouse, 1:1000, Abcam ab78078), NeuN (mouse, 1:1000, Novus Biologicals NBP1-92693), GFAP (chicken, 1:1000, Novus Biologicals NBP1-05198), GFP (goat 1:500, Rockland, 600-101-215)

Techniques: Staining, Virus, Immunohistochemistry, Marker