ace2  (R&D Systems)

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet: (A) Enrichment analysis of the expression levels of interferon gamma signaling genes in the indicated cell lines using publicly available RNA-seq data sets. (B) Comparison of the expression levels of ACE2 protein (x-axis) and mRNAs in the interferon gamma signaling pathway (y-axis) in the indicated cell lines. (C) UMAP analysis of cell surface protein expression in the indicated cell lines. (D) RT-qPCR analysis of ACE2 mRNA expression in the indicated cell lines. (E) Immunoblot analysis of ACE2 and β-actin expression in the indicated cell lines. (F) Cells were inoculated with SARS-CoV-2 (USA-WA1-2020) bearing WT S or S E484D or B.1.526 SARS-CoV-2 variant bearing S-E484K substitution at an MOI of 1 i.u./cell. Cell-associated viral RNA levels was analyzed at 4 h and 72 h post-infection by RT-qPCR from n=3 independent replicates. Data show the mean, error bars show the SD.

Article Snippet: Antibodies were used at the following dilutions: β-actin (Santa Cruz, SC-8432, 1:1000; Sigma #A5316, 1:5000), anti-HA (Biolegend, clone 16B12, 1:2000), ACE2 (R&D Systems #AF933, 1:200).

Techniques: Expressing, RNA Sequencing, Comparison, Quantitative RT-PCR, Western Blot, Variant Assay, Infection

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet: (A) H522-ACE2 KO, H661 and H661-ACE2 KO cells were inoculated with SARS-CoV-2-mNG S E484D at an MOI of 1. RT-qPCR analysis of cell-associated viral RNA at 4 and 72 hpi is shown. (B) WT H661 and four different H661 ACE2 KO clonal cells were inoculated with SARS-CoV-2-mNG S E484D at an MOI of 1. RT-qPCR for cell-associated viral RNA at 72 hpi is shown. (C) H522 and H661 WT cells pre-treated with 50 mM ammonium chloride for 2 h and inoculated with SARS-CoV-2-mNG S E484D at an MOI of 1. At 24 hpi, ammonium chloride was removed, and cells were replenished with fresh media. RT-qPCR analysis of cell-associated SARS-CoV-2 RNA at 48 hpi is shown. (D) H522 and H661 cells were pre-treated with a combination of heparinase I, II and III for 90 min. Cells were subsequently inoculated with SARS-CoV-2-mNG S E484D at an MOI of 0.1. RT-qPCR for cell-associated SARS-CoV-2 RNA at 72 hpi is shown. (E, F) H522 (E) and H661 cells (F) were infected with SARS-CoV-2-mNG S E484D (MOI of 0.1) in the presence of mAbs. Amount of cell-associated vRNA was quantified as above. All data show the mean from three independent biological replicates with error bars displaying the SEM. (nonsignificant (ns); ** P < 0.01; *** P < 0.001; **** P < 0.0001 by two-tailed unpaired t -test or one-way ANOVA with Dunnett’s correction for multiple comparisons).

Article Snippet: Antibodies were used at the following dilutions: β-actin (Santa Cruz, SC-8432, 1:1000; Sigma #A5316, 1:5000), anti-HA (Biolegend, clone 16B12, 1:2000), ACE2 (R&D Systems #AF933, 1:200).

Techniques: Quantitative RT-PCR, Infection, Two Tailed Test

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet: (A) H522 and H661 TMEM106B KO cells stably transduced with an empty vector (EV), full length TMEM106B (FL), TMEM106BΔCTD, TMEM106BΔNTD as well as H522 cells stably expressing ACE2 were inoculated with SARS-CoV-2-mNG S E484D or WT SARS-CoV-2-mNG at an MOI of 1 at 16°C. At 1 hpi, cells were fixed and analyzed for viral RNA (green) using RNAScope as detailed in Materials and Methods. Nuclear DAPI staining is shown in blue. (B) Quantification of the data shown in A. For S E484D infections, all data was compared to EV, for S-WT infections H522-ACE2 OE data was compared to H522-TMEM106BΔNTD (* P < 0.05; **** P < 0.0001 by two-tailed unpaired t -test or one-way ANOVA with Dunnett’s correction for multiple comparisons). Scale bar=10μM.

Article Snippet: Antibodies were used at the following dilutions: β-actin (Santa Cruz, SC-8432, 1:1000; Sigma #A5316, 1:5000), anti-HA (Biolegend, clone 16B12, 1:2000), ACE2 (R&D Systems #AF933, 1:200).

Techniques: Stable Transfection, Transduction, Plasmid Preparation, Expressing, RNAscope, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet: (A) H522 and H661 WT cells were inoculated with WT VSV-GFP-SARS-CoV-2 S (WT) or a derivative bearing the S E484D substitution at an MOI of 1. GFP positive cells were enumerated by flow cytometry at 48 hpi. (B) VSV-GFP-SARS-CoV-2 S E484D was serially passaged in H522 cells for 8 rounds as explained in Materials and Methods. Aliquots of virus from each passage was titered on H522 and H522-TMEM106B KO cells using 1, 5, 25 or 125 µl of the inoculum. Data show the percentage of GFP positive cells at the indicated inoculum for all 8 passages. (C) Aliquots of virus from (B) was titered on H661 cells. (D) Five different virus clones were plaque purified from Passage 7 and sequenced. Table shows the observed substitutions and their location within S. (E-G) H522-ACE2 ( E ), H522 ( F ), and H661 ( G ) cells were inoculated with parental VSV-GFP-SARS-CoV-2 S E484D or the five different plaque purified virus clones from (D) at an MOI of 0.1. H522-ACE2 cells were fixed at 16 hpi, and H522 and H661 were fixed at 48 hpi for enumeration of GFP positive cells by flow cytometry. Data show the mean from three independent experiments, error bars show the SEM. For D, E, and F, all five clones were compared to the parental virus (nonsignificant (ns), * P < 0.05 by one-way ANOVA with Dunnett’s correction for multiple comparisons).

Article Snippet: Antibodies were used at the following dilutions: β-actin (Santa Cruz, SC-8432, 1:1000; Sigma #A5316, 1:5000), anti-HA (Biolegend, clone 16B12, 1:2000), ACE2 (R&D Systems #AF933, 1:200).

Techniques: Flow Cytometry, Virus, Clone Assay, Purification

Journal: Frontiers in Pharmacology

Article Title: Bromhexine inhibits SARS-CoV-2 Omicron and variant pseudovirus infection via ACE2-targeted mechanisms

doi: 10.3389/fphar.2025.1745277

Figure Lengend Snippet: Immunofluorescence and Western blot analysis of ACE2 in HEK-293 and HEK-293/ACE2 cell lines. (A,C) Immunofluorescence localization of ACE2 proteins (red fluorescence). (B,D) Immunostaining images with primary antibodies omitted (control). All images show cell nuclei stained with DAPI (blue fluorescence). The scale bar represents 20 µm. (E) Representative immunoblots for ACE2 (90 kDa) and Actin (42 kDa) as a loading control are shown for HEK-293 cells and the stable cell line overexpressing human ACE2 (HEK-293/ACE2). (F) The relative abundance of ACE2 protein levels is expressed as the ratio of ACE2 to Actin band intensities. Data are shown as mean ± SEM from three independent experiments.

Article Snippet: Briefly, cells were seeded on coverslips, fixed with 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) for 20 min at room temperature, and permeabilized with 2% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 for 30 min. After blocking, cells were incubated overnight at 4 °C with mouse monoclonal primary antibodies against ACE2 (1:50 dilution, sc-390851; Santa Cruz Biotechnology, Dallas, TX, United States).

Techniques: Immunofluorescence, Western Blot, Fluorescence, Immunostaining, Control, Staining, Stable Transfection

Journal: Frontiers in Pharmacology

Article Title: Bromhexine inhibits SARS-CoV-2 Omicron and variant pseudovirus infection via ACE2-targeted mechanisms

doi: 10.3389/fphar.2025.1745277

Figure Lengend Snippet: Effect of bromhexine on SARS-CoV-2 Omicron pseudovirus infectivity in HEK-293/ACE2 cells. (A,C,E) Representative fluorescence microscopy images of cells infected with Omicron pseudovirus treated with 1, 10, and 100 µM bromhexine, respectively. (B) Positive control (Omicron pseudovirus infection without bromhexine). (D) Negative control (cells without Omicron pseudovirus infection or bromhexine). (F) Quantitative analysis of Omicron pseudovirus infection in HEK-293/ACE2 cells, based on the percentage of GFP-positive cells after infection. GFP expression indicates successful pseudovirus entry. Data are presented as means ± SEM ( n = 4) from at least three different experiments. An asterisk (*) indicates statistically significant differences ( p < 0.05) compared to the positive control, determined by one-way ANOVA with post hoc Tukey HSD test.

Article Snippet: Briefly, cells were seeded on coverslips, fixed with 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) for 20 min at room temperature, and permeabilized with 2% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 for 30 min. After blocking, cells were incubated overnight at 4 °C with mouse monoclonal primary antibodies against ACE2 (1:50 dilution, sc-390851; Santa Cruz Biotechnology, Dallas, TX, United States).

Techniques: Infection, Fluorescence, Microscopy, Positive Control, Negative Control, Expressing

Journal: Frontiers in Pharmacology

Article Title: Bromhexine inhibits SARS-CoV-2 Omicron and variant pseudovirus infection via ACE2-targeted mechanisms

doi: 10.3389/fphar.2025.1745277

Figure Lengend Snippet: Luciferase activity and IC 50 determination on HEK-293/ACE2 cells. (A) Infectivity of Omicron pseudoviruses was assessed by measuring luciferase activity in relative luminescence units (RLUs) after infecting cells with the viruses. Data are shown as means ± SEM ( n = 4). An asterisk (*) indicates statistically significant differences ( p < 0.05) determined by one-way ANOVA with post hoc Tukey HSD test. (B) Dose-response curve used to determine the half-maximal inhibitory concentration (IC 50 ) of bromhexine in HEK-293/ACE2 cells infected with Omicron pseudovirus, with an IC 50 of 17.3 ± 0.9 µM. Results are presented as means ± SEM ( n = 4). Curves are fitted to a 4-parameter logistic model and generated using the average of fitted parameters from individual experiments.

Article Snippet: Briefly, cells were seeded on coverslips, fixed with 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) for 20 min at room temperature, and permeabilized with 2% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 for 30 min. After blocking, cells were incubated overnight at 4 °C with mouse monoclonal primary antibodies against ACE2 (1:50 dilution, sc-390851; Santa Cruz Biotechnology, Dallas, TX, United States).

Techniques: Luciferase, Activity Assay, Infection, Concentration Assay, Generated

Journal: Frontiers in Pharmacology

Article Title: Bromhexine inhibits SARS-CoV-2 Omicron and variant pseudovirus infection via ACE2-targeted mechanisms

doi: 10.3389/fphar.2025.1745277

Figure Lengend Snippet: Reduction in infectivity of SARS-CoV-2 pseudovirus variants in HEK-293/ACE2 cells treated with bromhexine. Infectivity of pseudoviruses representing Alpha, Beta, and Delta SARS-CoV-2 variants was measured by luciferase activity, expressed in relative luminescence units (RLUs), 48 h after treatment with 40 μM bromhexine or a vehicle (control). The cells were infected with pseudoviruses engineered to express each variant. Data are shown as means ± SEM ( n = 4). An asterisk (*) indicates statistically significant differences ( p < 0.05) compared to the control group, determined by two-way ANOVA followed by post hoc Tukey HSD test.

Article Snippet: Briefly, cells were seeded on coverslips, fixed with 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) for 20 min at room temperature, and permeabilized with 2% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 for 30 min. After blocking, cells were incubated overnight at 4 °C with mouse monoclonal primary antibodies against ACE2 (1:50 dilution, sc-390851; Santa Cruz Biotechnology, Dallas, TX, United States).

Techniques: Infection, Luciferase, Activity Assay, Control, Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: Ultrastructural Features, Immune Response, and Junctional Proteins in the Seminiferous Epithelium of SARS-CoV-2-Infected Mice

doi: 10.3390/ijms27020691

Figure Lengend Snippet: ( A – D ): Photomicrographs of testicular sections of animals showing double immunofluorescence for hACE2 and spike in animals from CG and IG ( A – D ). Nuclear staining with DAPI. In ( A – D ), sections of seminiferous tubules at stages VII–VIII show hACE2 immunoexpression (arrows) in both groups. In ( B – D ), in addition to hACE2 (arrows), spike immunolabeling (arrowheads) is observed throughout the seminiferous epithelium of IG. In ( C , D ), enhanced spike and hACE2 immunolabeling is observed in damaged regions of the seminiferous epithelium, which show reduced height (double headed arrow) and intraepithelial spaces due to loss of germ cells (*). ( E – G ) : Photomicrographs of testicular sections of animals showing immunofluorescence for nucleocapsid protein in animals from IG. Nuclear staining with DAPI. In ( E ), seminiferous tubules at stages VII–VIII show nucleocapsid immunolabeling (arrows) in Sertoli cells (inset 1), round spermatids (inset 2), and flagellum of elongate spermatids (inset 3). In ( F , G ), nucleocapsid immunoreaction is observed in Sertoli cells’ cytoplasm and elongate spermatids (arrows) of IG. (SC) Sertoli cell nucleus. SC nucleolus (arrowheads). ( H ): A weak angiotensin II signal is observed in CG when compared to a strong signal in IG. The β-tubulin signal is observed in both groups. A significant increase in angiotensin II optical density (OD) is observed in IG when compared to CG.

Article Snippet: All sections were incubated in 2% BSA for 30 min, and incubated at 4 °C overnight with the following primary antibodies: mouse anti-hACE2 monoclonal antibody (RRID: AB_2861379, 1:500, Santa Cruz Biotechnology, Dallas, TX, USA, SC-73668, lot: #G1222), rabbit anti-SARS-CoV-2 spike protein S1 recombinant monoclonal antibody (RRID: AB_2866477, 1:250, Invitrogen, Carlsbad, CA, USA, MA5-36247, lot: XG3635472), rabbit anti-SARS-CoV-2 nucleocapsid protein monoclonal antibody (1:3000; EPR24334-118; Abcam, Cambridge, UK; ab271180), rabbit anti-Ki-67 monoclonal IgG antibody (1:200; Abcam, Cambridge, UK; ab16667), and rabbit anti-IFN-γ polyclonal IgG antibody (1:300, Invitrogen, cat. 95560, lot: XH3666559); mouse anti-TNF-α monoclonal IgG [52B83] antibody (1:200, Abcam, Cambridge, UK; ab1793, lot:GR3446230), rabbit anti-iNOS recombinant polyclonal IgG [RM1017] antibody (1;1500; Abcam, Cambridge, UK; ab283655, lot:GR3436095-8), mouse anti-connexin 43 monoclonal antibody (RRID: AB_10707826, 1:200; Santa Cruz Biotechnology; sc-271837), and rabbit anti-NF-kB p65 polyclonal antibody ab31481 (1:200; Abcam, Cambridge, UK; ab31481).

Techniques: Immunofluorescence, Staining, Immunolabeling

Journal: Cell Death Discovery

Article Title: Modelling severe COVID-19 in TLR3-mutated hiPSCs-derived lung organoids

doi: 10.1038/s41420-025-02936-5

Figure Lengend Snippet: A Phase–contrast microscopy of alveolospheres embedded in 3D Matrigel at day 51 of culture. Scale bar 200 µm. B Haematoxylin and eosin-stained hLORG cross-section showing the typical epithelial morphology. Scale bar 50 µm. C–E ACE2, SFTPC protein and gene expression in hLORGs at day 51 of differentiation by immunohistochemistry and qPCR analysis. F FOXA2 protein expression in hLORGs at day 51 of differentiation by immunohistochemistry. Scale bar 50 µm. G DPP4 protein expression in hLORGs at day 51 of differentiation by immunofluorescence. Scale bar 50 µm. H , I Immunofluorescence images show the overall actin (red) and α-tubulin distribution (green) of an hLORG displaying a prominent epithelial structure (nuclei, blue). Scale bar 50 µm and 25 µm, respectively.

Article Snippet: Immunohistochemistry was performed on the organoids fixed in 4% formalin (Spitalieri et al.), using goat anti-ACE2 antibody (1:60, AF 933, R & D Systems, Minneapolis, Minnesota, MN, USA) and rabbit anti-SFTPC (1:1000, ab 3786, Chemicon, Temecula, California, CA, USA).

Techniques: Microscopy, Staining, Gene Expression, Immunohistochemistry, Expressing, Immunofluorescence