Journal: International Journal of Molecular Sciences

Article Title: Molecular Mechanisms of Endothelialitis in SARS-CoV-2 Infection: Evidence for VE-Cadherin Cleavage by ACE2

doi: 10.3390/ijms241512525

Figure Lengend Snippet: The extracellular domain of VE-cadherin is a substrate for the ectopeptidase ACE2: ( a ) VE-cadherin sequence. The yellow color highlights the amino acid sequence of the extracellular domain of the protein. The putative consensus site for the ACE2 cleavage is highlighted in red. ( b ) Representative western blot showing the human recombinant ACE2 (hrACE2) analyzed with the antibody directed against ACE2 extracellular domain. The positions of molecular mass standards (in kDa) are shown at the left ( c , d ). Representative western blots of VE-cadherin extracellular domain incubated overnight at room temperature with increasing concentrations of hrACE2 (0.1 to 4 ng/mL) in 50 mM 2-(N-Morpholino)-ethane sulfonic acid (MES), 300 mM NaCl, 10 μM ZnCL2, 0.01% Brig 35, pH 6.5). The samples were analyzed by SDS-PAGE and immunoblotting. The immunoreactive band was detected with the monoclonal anti VE-cadherin antibody (BV9) or the rabbit polyclonal anti-EC1 antibody (anti EC1). ( e , f ) Densitometric analysis of the 90 kDa band using ImageJ software showed a dose-dependent decrease in VE-cadherin band intensity. This experiment was performed four times (n = 4) under similar conditions with comparable results.

Article Snippet: Monoclonal mouse anti-human ACE2 Antibody (MAB933) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Sequencing, Western Blot, Recombinant, Incubation, SDS Page, Software

Journal: International Journal of Molecular Sciences

Article Title: Molecular Mechanisms of Endothelialitis in SARS-CoV-2 Infection: Evidence for VE-Cadherin Cleavage by ACE2

doi: 10.3390/ijms241512525

Figure Lengend Snippet: Analysis of circulating ACE2 in blood from patients with SARS-CoV-2 infection. ( a ) Blood samples from COVID-19 patients were analyzed by SDS-PAGE and immunoblotting. Blood samples were diluted serially from 1 to 10 followed by a dilution of 1 to 2.5. 15 μL of sample preparations were loaded onto an SDS-PAGE and then transferred onto a nitrocellulose membrane. Proteins on the blots were visualized by Ponceau staining. The positions of molecular mass standards (in kDa) are shown at the left. The membranes were incubated with the anti-ACE2 antibody (5 μg/mL) followed by incubation with the anti-mouse peroxidase antibody. The arrow on the left-hand side shows the immunoreactive band corresponding to cACE2. ( b ) Densitometry analysis of the immunoreactive band corresponding to cACE2 to compare the groups (n = 4 for mild infection and n = 5 for severe infection). Error bars represent mean ± SE of means. p values from analysis of variance were assessed using the Mann-Whitney test (* p < 0.05). These experiments were repeated at least four times in a similar configuration.

Article Snippet: Monoclonal mouse anti-human ACE2 Antibody (MAB933) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Infection, SDS Page, Western Blot, Staining, Incubation, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Molecular Mechanisms of Endothelialitis in SARS-CoV-2 Infection: Evidence for VE-Cadherin Cleavage by ACE2

doi: 10.3390/ijms241512525

Figure Lengend Snippet: Proposed model of Pulmonary Endothelial dysfunction in COVID patients: In the pulmonary environment, the ECs are surrounded by pneumocytes (P1, and P2) and macrophages that are responsible for the cytokine storm. ACE2 is present on ECs as a transmembrane protein whose catalytic site is outside the cells (ectopeptidase). ACE2 can be cleaved by ADAM 17 and leads to the generation of a circulating active form of ACE2. VE-cadherin is a transmembrane protein exclusively expressed in ECs, which can be subjected to post-translational modifications including tyrosine phosphorylation upon cytokine challenge. This covalent modification will lead to a conformational change in the protein structure, which presents a high sensitivity to proteases. Thus, the ACE2 enzyme will act directly to its potential site of cleavage generating the fragments of VE-cadherin seen in the patient’s blood.

Article Snippet: Monoclonal mouse anti-human ACE2 Antibody (MAB933) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Modification

Journal: medRxiv

Article Title: Angiotensin I-Converting Enzyme type 2 expression is increased in pancreatic islets of type 2 diabetic donors

doi: 10.1101/2023.06.25.23291752

Figure Lengend Snippet: ( A ) Representative confocal images of FFPE pancreatic section from nondiabetic (ND) (case 13) and T2D donor (case 38). Pancreatic sections were stained with anti-ACE2 Ab MAB933 (green, panels a and e) and the merge with DAPI (white, nuclei) (panels b and f) showing the expression of ACE2-MAB933 in a pancreatic islet; pancreatic sections were stained with anti-ACE2 Ab ab15348 (blue, panels c and g) and the merge with DAPI (white, nuclei) (panel d and h) showed the expression of ACE2-ab15348 in a pancreatic islet. ( B ) Representative confocal images of FFPE pancreatic section derived from a nondiabetic (ND) (case 2) and a T2D donor (case 32). Pancreatic sections were stained for insulin (INS, red, panels a and f), ACE2-MAB933 (green, panels b and g) and ACE2-ab15348 (blue, panels c and h). Colocalization between ACE2-MAB933 and insulin is showed in yellow (panels d and i), while colocalization between ACE2-ab15348 and insulin is reported in magenta (panels e and j). Signal intensity analysis measured with anti-ACE2-MAB933 ( C ) and with anti-ACE2- ab15348 ( D ) antibody in non diabetic and T2D pancreatic sections; values are shown as fluorescence intensity of each islet detected (ND=556 islets; T2D=526 islets) reported as the sum of gray-scale values for each pixel normalized for the islets area (ROI, mm 2 ). (E-F) Colocalization rate analysis between ACE2-MAB933-Insulin ( E ) and ACE2-ab15348-insulin ( F ). Values are shown the colocalization rate (ROI, mm 2 ). * p < 0.05, ** p < 0.01, *** p < 0.001, non-parametric Mann-Whitney U test, performed after checking normality with the Kolmogorov-Smirnov normality test.

Article Snippet: To cross-validate the ACE2 staining results, we used two different anti-ACE2 antibodies: ( i ) a monoclonal mouse IgG2a anti-human ACE2 (R&D, MAB933), whose specificity was confirmed through an isotype primary antibody staining ( ESM ) and ( ii ) a rabbit polyclonal anti-ACE2 (Abcam, Ab15348), whose specificity was tested through a peptide competition assay and subsequent staining in ND donors pancreatic sections ( ESM ).

Techniques: Staining, Expressing, Derivative Assay, Fluorescence, MANN-WHITNEY

Journal: medRxiv

Article Title: Angiotensin I-Converting Enzyme type 2 expression is increased in pancreatic islets of type 2 diabetic donors

doi: 10.1101/2023.06.25.23291752

Figure Lengend Snippet: ( A ) Representative confocal images of FFPE pancreatic section from nondiabetic (ND) (case 13) and T2D donor (case 38). Pancreatic sections were stained with anti-ACE2 Ab MAB933 (green, panels a and e) and the merge with DAPI (white, nuclei) (panels b and f) showing the expression of ACE2-MAB933 in a pancreatic islet; pancreatic sections were stained with anti-ACE2 Ab ab15348 (blue, panels c and g) and the merge with DAPI (white, nuclei) (panel d and h) showed the expression of ACE2-ab15348 in a pancreatic islet. ( B ) Representative confocal images of FFPE pancreatic section derived from a nondiabetic (ND) (case 2) and a T2D donor (case 32). Pancreatic sections were stained for insulin (INS, red, panels a and f), ACE2-MAB933 (green, panels b and g) and ACE2-ab15348 (blue, panels c and h). Colocalization between ACE2-MAB933 and insulin is showed in yellow (panels d and i), while colocalization between ACE2-ab15348 and insulin is reported in magenta (panels e and j). Signal intensity analysis measured with anti-ACE2-MAB933 ( C ) and with anti-ACE2- ab15348 ( D ) antibody in non diabetic and T2D pancreatic sections; values are shown as fluorescence intensity of each islet detected (ND=556 islets; T2D=526 islets) reported as the sum of gray-scale values for each pixel normalized for the islets area (ROI, mm 2 ). (E-F) Colocalization rate analysis between ACE2-MAB933-Insulin ( E ) and ACE2-ab15348-insulin ( F ). Values are shown the colocalization rate (ROI, mm 2 ). * p < 0.05, ** p < 0.01, *** p < 0.001, non-parametric Mann-Whitney U test, performed after checking normality with the Kolmogorov-Smirnov normality test.

Article Snippet: After 3 washes in PBS 1X, the sections were incubated with monoclonal mouse anti-human ACE2 (cat. MAB933 - R&D System, Minneapolis, MS, USA) (final concentration: 15 µg/ml) for 1 hour at RT.

Techniques: Staining, Expressing, Derivative Assay, Fluorescence, MANN-WHITNEY

Journal: medRxiv

Article Title: Angiotensin I-Converting Enzyme type 2 expression is increased in pancreatic islets of type 2 diabetic donors

doi: 10.1101/2023.06.25.23291752

Figure Lengend Snippet: ( A ) Representative confocal images of FFPE pancreatic section from nondiabetic (ND) (case 13) and T2D donor (case 38). Pancreatic sections were stained with anti-ACE2 Ab MAB933 (green, panels a and e) and the merge with DAPI (white, nuclei) (panels b and f) showing the expression of ACE2-MAB933 in a pancreatic islet; pancreatic sections were stained with anti-ACE2 Ab ab15348 (blue, panels c and g) and the merge with DAPI (white, nuclei) (panel d and h) showed the expression of ACE2-ab15348 in a pancreatic islet. ( B ) Representative confocal images of FFPE pancreatic section derived from a nondiabetic (ND) (case 2) and a T2D donor (case 32). Pancreatic sections were stained for insulin (INS, red, panels a and f), ACE2-MAB933 (green, panels b and g) and ACE2-ab15348 (blue, panels c and h). Colocalization between ACE2-MAB933 and insulin is showed in yellow (panels d and i), while colocalization between ACE2-ab15348 and insulin is reported in magenta (panels e and j). Signal intensity analysis measured with anti-ACE2-MAB933 ( C ) and with anti-ACE2- ab15348 ( D ) antibody in non diabetic and T2D pancreatic sections; values are shown as fluorescence intensity of each islet detected (ND=556 islets; T2D=526 islets) reported as the sum of gray-scale values for each pixel normalized for the islets area (ROI, mm 2 ). (E-F) Colocalization rate analysis between ACE2-MAB933-Insulin ( E ) and ACE2-ab15348-insulin ( F ). Values are shown the colocalization rate (ROI, mm 2 ). * p < 0.05, ** p < 0.01, *** p < 0.001, non-parametric Mann-Whitney U test, performed after checking normality with the Kolmogorov-Smirnov normality test.

Article Snippet: Then, sections were incubated with primary antibody monoclonal mouse anti-human ACE2 (cat. MAB933 - R&;D System, Minneapolis, MS, USA) (final concentration: 15 μg/ml) in PBS 1X supplemented with 3% BSA and primary antibody polyclonal rabbit anti-human ACE2 (cat. ab15348 - Abcam, Cambridge, UK) (final concentration: 0,5 μg/ml) in PBS 1X supplemented with 3% BSA, overnight at +4°C.

Techniques: Staining, Expressing, Derivative Assay, Fluorescence, MANN-WHITNEY

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: The immunohistochemistry was performed on 200 sections of each fetal brain and 20 sections of adult hippocampus using as primary antibodies: rabbit polyclonal anti-ACE2 (Abcam Cat# ab15348, RRID:AB_301861; 1/500); rabbit polyclonal anti-ACE2 (Sigma-Aldrich Cat#HPA000288, RRID:AB_1078160; 1/500), mouse monoclonal anti-ACE2 (R&D Systems Cat# MAB933, RRID:AB_2223153; 1/500), rat monoclonal anti-GFAP (Millipore Cat# 345,860-100UG, RRID:AB_10685458; 1/200); mouse monoclonal anti-Neuropilin-1 (Invitrogen Cat# 14-3042-82, RRID:AB_2572873 ; 1/200 ), goat polyclonal anti-Doublecortin (Santa Cruz Biotechnology Cat# sc-8066 , RRID:AB_2088494; 1/100); and rabbit polyclonal anti-TBR2 (Abcam Cat# ab23345, RRID:AB_778267; 1/200).

Techniques: 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: 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: The immunohistochemistry was performed on 200 sections of each fetal brain and 20 sections of adult hippocampus using as primary antibodies: rabbit polyclonal anti-ACE2 (Abcam Cat# ab15348, RRID:AB_301861; 1/500); rabbit polyclonal anti-ACE2 (Sigma-Aldrich Cat#HPA000288, RRID:AB_1078160; 1/500), mouse monoclonal anti-ACE2 (R&D Systems Cat# MAB933, RRID:AB_2223153; 1/500), rat monoclonal anti-GFAP (Millipore Cat# 345,860-100UG, RRID:AB_10685458; 1/200); mouse monoclonal anti-Neuropilin-1 (Invitrogen Cat# 14-3042-82, RRID:AB_2572873 ; 1/200 ), goat polyclonal anti-Doublecortin (Santa Cruz Biotechnology Cat# sc-8066 , RRID:AB_2088494; 1/100); and rabbit polyclonal anti-TBR2 (Abcam Cat# ab23345, RRID:AB_778267; 1/200).

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: The immunohistochemistry was performed on 200 sections of each fetal brain and 20 sections of adult hippocampus using as primary antibodies: rabbit polyclonal anti-ACE2 (Abcam Cat# ab15348, RRID:AB_301861; 1/500); rabbit polyclonal anti-ACE2 (Sigma-Aldrich Cat#HPA000288, RRID:AB_1078160; 1/500), mouse monoclonal anti-ACE2 (R&D Systems Cat# MAB933, RRID:AB_2223153; 1/500), rat monoclonal anti-GFAP (Millipore Cat# 345,860-100UG, RRID:AB_10685458; 1/200); mouse monoclonal anti-Neuropilin-1 (Invitrogen Cat# 14-3042-82, RRID:AB_2572873 ; 1/200 ), goat polyclonal anti-Doublecortin (Santa Cruz Biotechnology Cat# sc-8066 , RRID:AB_2088494; 1/100); and rabbit polyclonal anti-TBR2 (Abcam Cat# ab23345, RRID:AB_778267; 1/200).

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: The immunohistochemistry was performed on 200 sections of each fetal brain and 20 sections of adult hippocampus using as primary antibodies: rabbit polyclonal anti-ACE2 (Abcam Cat# ab15348, RRID:AB_301861; 1/500); rabbit polyclonal anti-ACE2 (Sigma-Aldrich Cat#HPA000288, RRID:AB_1078160; 1/500), mouse monoclonal anti-ACE2 (R&D Systems Cat# MAB933, RRID:AB_2223153; 1/500), rat monoclonal anti-GFAP (Millipore Cat# 345,860-100UG, RRID:AB_10685458; 1/200); mouse monoclonal anti-Neuropilin-1 (Invitrogen Cat# 14-3042-82, RRID:AB_2572873 ; 1/200 ), goat polyclonal anti-Doublecortin (Santa Cruz Biotechnology Cat# sc-8066 , RRID:AB_2088494; 1/100); and rabbit polyclonal anti-TBR2 (Abcam Cat# ab23345, RRID:AB_778267; 1/200).

Techniques: 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: The immunohistochemistry was performed on 200 sections of each fetal brain and 20 sections of adult hippocampus using as primary antibodies: rabbit polyclonal anti-ACE2 (Abcam Cat# ab15348, RRID:AB_301861; 1/500); rabbit polyclonal anti-ACE2 (Sigma-Aldrich Cat#HPA000288, RRID:AB_1078160; 1/500), mouse monoclonal anti-ACE2 (R&D Systems Cat# MAB933, RRID:AB_2223153; 1/500), rat monoclonal anti-GFAP (Millipore Cat# 345,860-100UG, RRID:AB_10685458; 1/200); mouse monoclonal anti-Neuropilin-1 (Invitrogen Cat# 14-3042-82, RRID:AB_2572873 ; 1/200 ), goat polyclonal anti-Doublecortin (Santa Cruz Biotechnology Cat# sc-8066 , RRID:AB_2088494; 1/100); and rabbit polyclonal anti-TBR2 (Abcam Cat# ab23345, RRID:AB_778267; 1/200).

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

Journal: The American Journal of Pathology

Article Title: Lung remodeling regions in long-term Covid-19 feature basal epithelial cell reprogramming

doi: 10.1016/j.ajpath.2023.02.005

Figure Lengend Snippet: Alveolar epithelial cell loss and viral receptor expression in lung remodeling regions in Covid-19. A, Immunostaining for KRT5 plus HT2-280, SFTPC, and PDPN with DAPI counterstaining in lung sections from Covid-19 patients and non-disease controls. Scale bar=100 μm. B, Quantitation of staining for conditions in (A). C , Immunostaining for ACE2 alone and with KRT5, HT2-280, SCGB1A1, or α-tubulin with DAPI counterstaining in lung sections for conditions in (C); immunostaining with rabbit anti-ACE-2 pAb for serial sections and mouse anti-ACE-2 mAb for co-staining sections. White dashed-line box indicates ACE-2 + α-tubulin + cells. Scale bar=50 μm. Data are representative of 5 patients and 5 control subjects per staining condition. Values represent mean and s.e.m; * P <0.05 (n=5 patients or subjects per group).

Article Snippet: Immunostaining was performed using the following primary antibodies: rabbit anti-ACE-2 polyclonal Ab (pAb) (ab65863, Abcam), mouse anti-ACE-2 mAb (clone 171606, R&D Systems), rabbit anti-KRT5 pAb (ab53121, Abcam) and mAb (clone EP1601Y, ab52635, Abcam), rabbit anti-AQP3 pAb (ab125219, Abcam), mouse anti-CD68 mAb (clone Kp-1, Sigma-Aldrich), rabbit anti-CD163 mAb (clone D6U1J, Cell Signaling), rabbit anti-CD31 mAb (clone EPR17259, Abcam), rabbit anti-CollagenIV (CollIV) mAb (clone EPR20966, Abcam), mouse anti-SCGB1A1 mAb (clone E-11, Santa Cruz), mouse anti-acetylated Tubulin (clone 6-11B-1, Sigma-Aldrich), mouse anti-MUC5AC mAb (clone 45M1, ThermoFisher Scientific and Santa Cruz Biotechnology), mouse anti-HT2-280 pAb (TB-27AHT2-280, Terrace Biotech), rabbit anti-surfactant protein C (SFTPC) pAb (ab90716, Abcam), rabbit anti-podoplanin (PDPN) mAb (clone EPR7072, Abcam), rabbit anti-MUC5B pAb (ab87276, Abcam), rabbit anti-Ki-67 mAb (clone D2H10, Cell Signaling), rabbit active (cleaved) caspase-3 mAb (clone 5A1E, Cell Signaling), and mouse anti-CXCL17 mAb (clone 422204, R&D Systems).

Techniques: Expressing, Immunostaining, Quantitation Assay, Staining