Journal: Science immunology

Article Title: TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes.

doi: 10.1126/sciimmunol.abc3582

Figure Lengend Snippet: Fig. 1. VSV-SARS-CoV-2 infects human small intestinal enteroids. (A) Mouse small intestinal cells were analyzed by scRNA-seq and resolved into 20 clusters based on gene expression profiles (left). Transcript levels of Cd26, Epcam, Cd44, Cd45, and Ace2 were indicated for different intestinal cell subsets. Clusters 10 and 18: intraepithelial lymphocytes; clusters 1, 2, 5, 8, 9, 17, 19, and 20: enterocytes; cluster 3: goblet cells; cluster 4: enteroendocrine cells; cluster 7: Tuft cells; cluster 11: crypt stem cells; cluster 12: Paneth cells. Each dot represents a single cell. Note that Ace2high cells are also positive for Cd26 and Epcam but negative for Cd44 and Cd45. (B) Human duodenum enteroids were cultured in the Transwell monolayer system using maintenance (MAINT) or differentiation (DIFF) conditions for 3 days. Monolayers were stained for ACE2 (red) and actin (phalloidin, white). Scale bars, 32 m. (C) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically infected with 1.5 × 105 plaque-forming units (PFU) of VSV-SARS-CoV-2 [multiplicity of infection (MOI) = 0.3] for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. p.i., post-infection. (D) Human duodenum enteroids in 3D Matrigel were cultured in maintenance (MAINT) medium or differentiation (DIFF) medium for 3 days and infected with 2.2 × 105 PFU of VSV-SARS-CoV-2 for 18 hours. Enteroids were stained for virus (green), actin (phalloidin, white), and nucleus (DAPI, blue). Scale bars, 50 m. (E) Same as (C) except that virus titers were mea- sured using a TCID50 assay instead of viral RNA levels by qPCR. (F) Same as (D) except that human ileum enteroids were used instead. Scale bars, 80 m. (G) Same as (D) except that human colon enteroids were used instead. Scale bars, 80 m. For all figures except (A), experiments were repeated at least three times with similar re- sults. (A) was performed once with small intestinal tissues pooled from three mice. Data are represented as mean ± SEM. Statistical significance is indicated (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Article Snippet: Samples were then stained with the following primary antibodies or fluorescent dyes: ACE2 (sc-390851 AF594, Santa Cruz Biotechnology), 4′,6-diamidino-2-phenylindole (DAPI) (P36962, Thermo Fisher Scientific), SARS-CoV-2 S [CR3022 human monoclonal antibody (58)], villin (sc-58897 AF488, Santa Cruz Biotechnology), and phalloidin (Alexa 647–conjugated, Thermo Fisher Scientific).

Techniques: Gene Expression, Cell Culture, Staining, Infection, Expressing, Quantitative RT-PCR, Virus, TCID50 Assay

Journal: Science immunology

Article Title: TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes.

doi: 10.1126/sciimmunol.abc3582

Figure Lengend Snippet: Fig. 2. VSV-SARS-CoV-2 and wild-type SARS-CoV-2 replicate in ACE2+ human mature enterocytes. (A) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically or basolaterally infected with 1.5 × 105 PFU of VSV-SARS-CoV-2 for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (B) Supernatants in both apical and basal chambers were collected from (A) and were subjected to a TCID50 assay to measure the amount of infectious virus. (C) Differentiated duodenum enteroids in monolayer were apically infected with 2.5 × 105 PFU of infectious SARS-CoV-2 virus (MOI = 0.5) for 8 hours. The expression of SARS-CoV-2 N was measured by RT-qPCR using a TaqMan assay and normalized to that of GAPDH. (D) Differentiated ileum enteroids in monolayer were apically or basolaterally infected with 2.5 × 105 PFU of infectious SARS-CoV-2 virus (MOI = 0.5) for 8 hours. The expression of SARS-CoV-2 N was measured by RT-qPCR using a TaqMan assay and normalized to that of GAPDH. (E) Same as (C) except that enteroids were fixed and stained for SARS-CoV-2 S (green), ACE2 (red), and nucleus (DAPI, blue). Scale bars, 32 m. SARS-CoV-2–infected ACE2-positive cells are enlarged in the inset (yellow box). (F) SARS-CoV-2–infected duodenum monolayers were imaged along the Z stacks and sectioned for YZ planes (top) and reconstructed for 3D images (bottom). For all figures except (C) to (E), ex- periments were repeated at least three times with similar results. (C) to (E) were performed twice with technical duplicates in each experiment. Data are represented as mean ± SEM. Statistical significance is indicated (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Article Snippet: Samples were then stained with the following primary antibodies or fluorescent dyes: ACE2 (sc-390851 AF594, Santa Cruz Biotechnology), 4′,6-diamidino-2-phenylindole (DAPI) (P36962, Thermo Fisher Scientific), SARS-CoV-2 S [CR3022 human monoclonal antibody (58)], villin (sc-58897 AF488, Santa Cruz Biotechnology), and phalloidin (Alexa 647–conjugated, Thermo Fisher Scientific).

Techniques: Cell Culture, Infection, Expressing, Quantitative RT-PCR, TCID50 Assay, Virus, TaqMan Assay, Staining

Journal: Science immunology

Article Title: TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes.

doi: 10.1126/sciimmunol.abc3582

Figure Lengend Snippet: Fig. 3. TMPRSS2 and TMPRSS4, but not ST14, mediate SARS-CoV-2 S–mediated entry. (A) Bulk RNA-seq results of intestine-specific serine protease expression in HEK293, Huh7.5, H1-Hela, and HT-29 cells and human ileum enteroids. (B) HEK293 cells were transfected with pcDNA3.1-V5-ACE2, DDP4, or ANPEP for 24 hours (left), or transfected with indicated plasmid combination for 24 hours (right), and infected with 1.5 × 105 PFU of VSV-SARS-CoV-2 for 24 hours. The ex- pression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (C) HEK293 cells stably expressing human ACE2 were transfected with SARS-CoV-2 S and TMPRSS2 or TMPRSS4 for 48 hours. Cells were treated with trypsin at 0.5 g/ml for 10 min. The levels of S and GAPDH were measured by Western blot. The intensity of bands was quantified by ImageJ and shown as percentage of the bottom band versus the top band in each lane. (D) HEK293 cells stably expressing human ACE2 were transfected with TMPRSS2 or TMPRSS4 for 24 hours, incubated with 5.8 × 105 PFU of VSV-SARS-CoV-2 on ice for 1 hour, washed with cold phosphate-buffered saline for three times, and shifted to 37°C for another hour. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (E) Wild-type (WT) or human ACE2-expressing HEK293 cells were transfected with SARS-CoV-2 S and GFP, with or without TMPRSS2 or TMPRSS4 for 24 hours. The red arrows highlight the formation of large syncytia. Scale bars, 100 m. For all figures except (A), experiments were repeated at least three times with similar results. RNA-seq in (A) was performed once with duplicate samples. Data are represented as mean ± SEM. Statistical sig- nificance is indicated (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Article Snippet: Samples were then stained with the following primary antibodies or fluorescent dyes: ACE2 (sc-390851 AF594, Santa Cruz Biotechnology), 4′,6-diamidino-2-phenylindole (DAPI) (P36962, Thermo Fisher Scientific), SARS-CoV-2 S [CR3022 human monoclonal antibody (58)], villin (sc-58897 AF488, Santa Cruz Biotechnology), and phalloidin (Alexa 647–conjugated, Thermo Fisher Scientific).

Techniques: RNA Sequencing, Expressing, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Stable Transfection, Western Blot, Incubation, Saline

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: ACE2 expression in human fetal hippocampus. A Image of a coronal section of the temporal lobe showing the hippocampal formation. Insert represents the picture showed in ( B ). B Picture showing the Hippocampus Fimbrial Angle (HFA) between the fimbria and the Ammon’s horn (CA) ventricular surfaces. Inserts represent the areas that are showed in C, D, and F. ACE2 immunopositive (ACE2 +) migratory cells were observed from the HFA ventricular epithelium (VE) to the subpial region (pial surface: ps), through a radial migratory stream of cells toward the dentate gyrus (DGMS). C High power photomicrograph of the HFA showing the distribution of ACE2 + cells in the ventricular epithelium (VE) and the subventricular zone (SVZ). D High power photomicrograph of ventricular surface showing ACE2 + cells in VE and the subventricular zone (SVZ) of proximal ( D ) side of the HFA. E Ventricular surface showing ACE2 immunopositive particles accumulate on the basal pole membrane of VE progenitors (arrows). F Picture showing ACE2 + migrating cells reaching the developing Dentate Gyrus (DG) at the pial surface (ps) of FDS. G High power photomicrographs of migratory ACE2 + cells reaching he ps of the DGMS. H – J High power pictures showing ACE2 expression in the advance processes of migrating cells: (arrows in J and H ), and cells around blood vessels (arrows in I ). K – M ACE2 expression in choroidal plexus cells. Both epithelial cells (ec) in the ventricular surface, and stromal pericytes (pc) around blood vessels (bv) were immunopositive (arrows). N ACE2 expression in DG ventricular epithelium and DGMS at the caudal pole of hippocampus (FC and IG). Scale bar: A 500 µm; B 300 µm; C , D 50 µm; E 10 µm; F 150 µm; G , N 100 µm; H–K 15 µm; L , M 5 µm. bv blood vessel, CA cornu ammonis, DGMS dentate gyrus migratory stream, ec epithelial cell, FDS fimbrio-dentate sulcus, Fi fimbria, HFA hippocampus fimbrial angle, pc pericytes, ps pial surface, SVZ sub-ventricular zone, st choroidal plexus stroma, VE ventricular Epithelium

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Expressing, Membrane

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: Migration routes of DG progenitors. A Picture of a coronal section of the temporal lobe showing GFAP expression in radial glia cells and fibers of the Sub-ventricular Zone (SVZ) and Migratory Stream (DGMS) of the Dentate Gyrus (DG), from the Hippocampus Fimbrial Angle (HFA) to the pial surface (ps). DGMS has been delimited by arrows. B High power picture of the DG Ventricular Epithelium (VE), Sub-ventricular Zone (SVZ) and intermediate zone (IZ). Arrows label GFAP positive fibers. C High power picture of ACE2-expressing cells (cells with DAB-nickel precipitate; arrows) following GFAP radial glia fibers of DG ventricular epithelium (VE; DAB precipitate; arrows head). D , E Cresyl violet staining showing the DG ventricular (VE) and sub-ventricular (SVZ) zones with perivascular accumulation of cells crossing the sub-ventricular withe matter (alveus) in the DG migratory stream (DGMS; arrows). F ACE2 expression in perivascular migrating cells (arrowheads) and pericytes (arrows) in the alveus. G Doublecortin expression in DG migrating neuronal precursors and neurons in DG molecular layer (DGML). ACE2 is expressed in migratory precursors of the DGMS (DAB-nickel precipitate; arrows) but not in DGML, single doublecortin expressing cells (DAB precipitate). Scale bar: A 200 µm; B 100 µm; C – E 50 µm; F 25 µm. bv blood vessel, DGH Dentate Gyrus Hilux, DGMS Dentate Gyrus Migratory Stream, DGML Dentate Gyrus Molecular Layer, HFA Hippocampus Fimbrial Angle, ps pial surface, VE ventricular epithelium

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Migration, Expressing, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: ACE2 + cells in DGMS are TBR2 + neural progenitors. A High power picture showing DG ventricular epithelium (VE) expressing ACE2 and TBR2 in the Hippocampal Fimbrial Angle (HFA). Double immunopositive, ACE2 and TBR2, cells are mainly localized in the basal surface of the VE (arrows) and in cells that are migrating into the subventricular zone (arrowhead). B Microphotograph showing the Dentate Gyrus Migratory Stream (DGMS) from the HFA to the pial surface (ps), where DGMS migratory cells follow dorsally to the Dentate Gyrus Hilux (DGH) or ventrally to Dentate Gyrus Molecular Layer (DGML). Insets localized the areas showed in ( A , C , and D ). C High power microphotograph showing ACE2 (DAB precipitate in the cytoplasm; arrowheads) and TBR2 (DAB-nickel precipitate in the nucleus; arrows) expression in ventricular and migratory cells. D High power of the zone represented in the inset of B showing double labeled ACE2 and TBR immunopositive cells in the DGMS (arrows). E Picture showing DG ventricular epithelium (VE) expressing ACE2 and TBR2 at the Hippocampal Fimbrial Angle (HFA) (small arrows) and in migrating cells in the SVZ and DGMS (arrowheads). Large arrows show ACE2 and TBR immunopositive cells migrating near to a blood vessel (bv). F Low power picture of an immunofluorescence processed section, showing the DGMS at the caudal pole of the hippocampus, where the DG turns dorsally to became the fasciola cinerea. Ventral and dorsal DGMS are delimited by arrows. G , H High power picture of immunofluorescence showing ACE2 immunolabeling in the cytoplasm (green) and TBR2 immunolabeling in the nucleus (reed) in DGMS cells. Scale bar: A , C , E , G 10 µm: B 200 µm; D , H 7 µm; F 300 µm. bv blood vessel, CA Ammon’s horn, DGH Dentate Gyrus Hilux, DGML Dentate Gyrus Molecular Layer, DGMS Dentate Gyrus Migratory Stream, HFA Hippocampus Fimbrial Angle, ps pial surface, SVZ sub-ventricular Zone, VE ventricular epithelium

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Expressing, Labeling, Immunofluorescence, Immunolabeling

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: ACE2 and neuropilin 1 (NP1) co-localize in the membrane of DGMS cells. A ACE2 immunofluorescent cells in the DGMS between sub-ventricular zone of the HFA and the ps (arrows). B High power confocal photomicrograph sowing the co-expression of ACE2 and NP1 in the membrane of migrating cells (arrows). The arrowhead shows the point where Z-stack projection have been reconstructed (view in the x and z plane), to demonstrate co-localization of ACE2 (reed dots) and NP1 (green/yellow dots) in the cellular membrane. C – E Microphotographs showing ACE2 and NP1 co-expression in DGMS cells. Inserts show the same cells with clear co-expression of ACE2 and NP1 in the membrane of leading processes (arrows). F – H DGMS cell showing strong co-expression of ACE2 and NP1 in the cellular membrane of leading processes (arrows). Scale bar: A : 250 µm; B 10 µ; C – E 20 µm; F – H 15 µm

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Membrane, Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: ACE2 expression in migrating neural crest progenitors. A , B Anti-ACE2 immunofluorescence showed expression of ACE2 in the cytoplasm of cultured dental derived NCP, with perinuclear accumulation. A’ Dental derived NCP express Nestin protein (red immunofluorescence). B’ Cells with polarized morphology ACE2 expression is stronger and accumulated in the cellular membrane of the progression pole (arrows). C – I Scratch-wound assay in confluent cultures. D 10 min after the scratch (t0h) cells at the wound edge strongly expressed ACE2 (arrows). E – I At 12–24 h (t12h, t24h) after the scratch, polarized elongated cells migrated into the wound with strong expression of ACE2 in the cellular membrane of advance processes (arrows) including in intercellular nanotubes (arrows head in H ). I Quantification of ACE2 expression by immunofluorescence intensity and intracellular distribution with the ImageJ tool for measuring corrected total cell fluorescence (CTCF). Data shown as mean ± S.D. The number of cells analyzed were: T0h (cytoplasm or membrane) n = 26, T12h (cytosol or membrane) n = 22. Comparisons between cytoplasm and cell membrane at T0h and at T12h were made with tests for paired samples (Wilcoxon signed-rank test) and comparisons between cytoplasm at T0h and T12h or cell membrane at T0h and T12h were made with test for independent samples (Mann–Whitney test for T0h test), using the software GraphPad Prism. a.u arbitrary units. Asterisks indicate p -value: **** p < 0.0001 ns not significant. J At 48 h after the scratch the wound was completely cellularized with increased expression of ACE2 in some cell membranes of neighboring cells at the place where the wound was performed (arrows)

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Expressing, Immunofluorescence, Cell Culture, Derivative Assay, Membrane, Scratch Wound Assay Assay, Fluorescence, MANN-WHITNEY, Software

Journal: Nature Communications

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

doi: 10.1038/s41467-020-17796-z

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

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

Techniques: Infection, Marker, Staining

Journal: Nature Communications

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

doi: 10.1038/s41467-020-17796-z

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

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

Techniques: Concentration Assay, Immunofluorescence