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: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Glycan profiles of the SARS-CoV-2 S1 and the ACE2 receptor. (A) Schematic diagram illustrating the process of preparing antibody-overlay lectin microarrays. (B, C) Scanned images were obtained for the analysis of glycopatterns from the SARS-CoV-2-S1 (B) and ACE2 (C). HEK293-expressing recombinant proteins of S1 and ACE2 were incubated with lectin microarrays. Subsequently, the microarrays were incubated with biotin-labeled primary antibodies and Cy3-labeled streptavidin. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II and STL), bisected and bi-antennary N-glycans (PHA-E), oligo-mannose type N-glycans (ConA and HHL), fucosylation (PSA and LCA) and α-2,3 linked sialic acid (MAL-II) were marked with white frames. (D, E) Analysis of glycopatterns on S1 (D) and ACE2 (E). The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of main types of glycans was calculated by diverging the sum of the NFIs of the lectins that recognized this type of glycan by the total NFIs of all lectins. Blue square: GlcNAc; yellow circle: galactose; yellow square: GalNAc; green circle: mannose; red triangle: fucose; purple diamond: sialic acid. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Glycoproteomics, Expressing, Recombinant, Incubation, Labeling, Binding Assay

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Role of N-glycans in the interaction between S1 and ACE2. (A) Schematic diagram illustrating the process of manufacturing the SRAS-CoV-2-related recombinant protein microarrays. (B, C) The N-glycans on S1 of SARS-CoV-2/1 and ACE2 were removed by PNGase F glycosidase. The roles of N-glycans in the interaction between the SARS-CoV-2-S1 /ACE2 (B) and the SARS-CoV-1-S1/ACE2 (C) were evaluated using protein microarrays. Statistical analysis of the relative fluorescence intensities was conducted by comparing the PNGase F-treated S1 and ACE2 to the intact glycosylated protein using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (D) MD simulation of the interaction between the trimeric S protein and ACE2. The distances between the N-glycosites and the center of the binding interface (represented by the green globule) within 50 Å were marked with red spheres. Other N-glycosites were marked with yellow spheres. (E) The interactions of glycans at specific sites and GRDs (marked with a red frame) may be involved in the binding of the S protein to ACE2. (F) MD simulated the interactions of glycans at specific sites and GRDs. The distances between the terminal glycans on these sites and the three GRDs on the ACE and S1 subunit were monitored during a 100 ns MD simulation. The distances of N546-GRD1, N322-GRD2, and N53-GRD2 fluctuated between 1 and 15 Å, while the distances of N343-GRD3 and N165-GRD3 fluctuated between 20 and 35 Å. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Recombinant, Fluorescence, Binding Assay

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: β1-4 galactosylated N-glycans of ACE2 mediated the binding of S1 of SARS-CoV-2 and its variants. (A) Molecular docking analysis of S1 and ACE2 with various saccharides. The potential binding capacities of S1 of SARS-CoV-2 (Wuhan-Hu-1 strain, wild type) and its variants (Delta and Omicron), as well as ACE2, to various saccharides were predicted by molecular docking analysis. The saccharides were listed in columns, S1 and ACE2 were listed in rows. The different binding abilities were represented by the values of binding free energy, which were indicated by the color of each square: red: high affinity, blue: low affinity, Xyl: xylose, Glc: glucose; Man: mannose; GlcNAc: N-acetylglucosamine, GalNAc: N-acetylgalactosamine; SA: sialic acid. (B) Validation of β1-4 galactosylation level in intact and de-β1-4galactosylated ACE2. After β1-4 galactosidase treatment, the level of β1-4 galactosylation on ACE2 was detected by lectin blotting of MAL-I. The protein level of ACE2 served as the control. (C) Scanning images of protein microarrays incubated with 1 μg of intact or de-β1-4galactosylated ACE2. (D) Effect of β1-4 galactosylation of ACE2 on the binding of S1 to ACE2. The relative fluorescence intensities were statistically analyzed by comparing the de-β1-4galactosylated ACE2 to intact ACE2 using an unpaired t test with Welch's correction. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Binding Assay, Biomarker Discovery, Control, Incubation, Fluorescence

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Evaluation of the ability of free saccharides to block S1 and ACE2 binding. (A, B) Scanning images of protein microarrays. ACE2 was mixed with GalNAc (A) or Galβ1-3GalNAc (B), and the inhibitory effect of saccharides was evaluated using protein microarrays. (C, D) Effect of GalNAc (C) and Galβ-1,3GalNAc (D) on the interaction between S1 of SARS-CoV-2/1 and ACE2. The binding signals were extracted, and the relative fluorescence intensities were compared with those of the controls using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated.

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Blocking Assay, Binding Assay, Fluorescence

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Evaluation of isolated glycoproteins for the inhibition of S1 and ACE2 binding. (A) The scanned image was obtained from the lectin microarray analysis of glycoproteins isolated from bovine milk. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II), bisected N-glycans (PHA-E), high-mannose glycans (ConA), fucosylation (AAL, PSA, and LCA), α2-3 linked sialic acid (MAL-II), and α2-6 linked sialic acid (SNA) were marked with white frames. (B) Analysis of glycopatterns on isolated glycoproteins. The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of galactosylated glycans was calculated by diverging the sum of the NFIs of the lectins that recognized Gal/GalNAc by the total NFIs. (C, D) Evaluation of the effect of intact and de-sialylated isolated glycoproteins on the interaction between S1 of SARS-CoV-2/1 and ACE2. The intact isolated glycoproteins (C) or de-sialylated isolated glycoproteins (D) were mixed with ACE2 and incubated with protein microarrays. The relative binding intensities of each group were compared with those of the control group, and any significant differences between groups were determined using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (E) Inhibition curves for intact isolated glycoproteins (upper) and de-sialylated isolated glycoproteins (lower). Four-parameter inhibition curves were generated, and the particular IC50 values for intact isolated glycoproteins and de-sialylated isolated glycoproteins were indicated in this graph. The data were obtained from three biological replicates and presented as the mean ± SD (error bars).

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Isolation, Inhibition, Binding Assay, Microarray, Glycoproteomics, Incubation, Control, Generated

Journal: mBio

Article Title: Evolutionary dynamics of heparan sulfate utilization by SARS-CoV-2

doi: 10.1128/mbio.01303-25

Figure Lengend Snippet: The Omicron variant infects HEK293T cells expressing low levels of endogenous ACE2. ( A ) Binding of WT (B lineage) or Omicron (BA.1) RBD fused to human IgG Fc (RBD-Fc), or anti-ACE2 Ab to mock, ACE2 KO, or ACE2-transfected (Tf) HEK293T cells. GMFI, geometric mean fluorescence intensity. ( B ) Titration of pseudoviruses bearing the SARS-CoV-2 D614G or BA.1 spike using VSV-G-Tf HEK293T cells. ( C ) Infection of mock, ACE2 KO, or ACE2-Tf HEK293T cells with D614G or BA.1 pseudovirus. RLU, relative luminescence units. ( D ) Infection of mock, ACE2 KO, or ACE2-Tf HEK293T cells with authentic SARS-CoV-2 WT or Omicron (BA.1.18) variant. Viral RNA in supernatants or cell lysates at 24 hours post-inoculation (hpi) is shown. Lysate RNA was normalized to Actb . Data are mean ± SEM of three to four technical replicates. Statistical analysis was performed using two-way analysis of variance (ANOVA) with Sidak’s multiple comparison tests in panels A and C and unpaired two-tailed Welch’s t -tests between WT and BA.1.18 in panel D ; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; ns, not significant. Data are representative of two to three independent experiments.

Article Snippet: Mouse anti-human ACE2 monoclonal antibody (mAb) (AC384, Adipogen), mouse anti-HS mAb (F58-10E4, Amzbio), mouse anti-CS mAb (CS-56, Sigma-Aldrich), rat anti-Flag-tag mAb (L5, BioLegend), mouse anti-human CD138 (syndecan-1) mAb (MI15, BioLegend), mouse anti-human glypican-4 mAb (961609, R&D Systems), mouse anti-human CD46 mAb (J4.48, Beckman), mouse anti-human CD59 mAb (p282[H19], BioLegend), mouse anti-human HLA class I mAb (W6/32, Institute of Development, Aging and Cancer, University of Tohoku), mouse anti-human CD51 (integrin αV) mAb (L230, Enzo), Alexa Fluor 555 goat anti-mouse IgM (Heavy chain) Ab (Thermo Scientific), allophycocyanin (APC)-conjugated goat anti-human IgG, Fcγ fragment specific Ab, APC-conjugated goat anti-mouse IgG, Fcγ fragment specific Ab, APC-conjugated goat anti-mouse IgM, μ chain specific Ab, APC-conjugated donkey anti-rat IgG (H + L) Ab, and APC-conjugated streptavidin (Jackson) were used.

Techniques: Variant Assay, Expressing, Binding Assay, Transfection, Fluorescence, Titration, Infection, Comparison, Two Tailed Test

Journal: mBio

Article Title: Evolutionary dynamics of heparan sulfate utilization by SARS-CoV-2

doi: 10.1128/mbio.01303-25

Figure Lengend Snippet: ACE2-independent binding of the Omicron RBD to cell surface HS. ( A ) CRISPR KO library screening scheme to identify Omicron RBD binding molecules expressed on HEK293T cells. ( B ) Binding of CRISPR KO library-transduced HEK293T cells with (red line) or without (shaded gray) Omicron RBD-Fc, before and after sorting. ( C ) Number of sgRNA sequences identified in Omicron RBD-Fc non-binding cells after sorting. Red: GAG-synthesis-related genes; black: other expressed genes; gray: non-expressed genes in HEK293T cells. ( D ) HS biosynthetic pathway highlighting SLC35B2 and B3GAT3. PAPS, 3'-phosphoadenosine-5'-phosphosulfate; Xyl, xylose; Gal, galactose; GlcNAc, N-acetylglucosamine; GlcA, glucuronic acid; IdoA, iduronic acid. ( E ) Binding of BA.1 RBD-Fc, anti-HS Ab, or anti-CS Ab to mock (black line), or SLC35B2 or B3GAT3 KO (red line) HEK293T cells. ( F ) Binding of WT or BA.1 RBD-Fc, PILRα-Fc, anti-HS Ab, or anti-CS Ab to ACE2 KO HEK293T cells pretreated with (+) or without (–) heparinase or chondroitinase. ( G ) Binding of WT or BA.1 RBD-Fc to HEK293T cells at different heparin concentrations. RBD-Fc binding was normalized to binding in the absence of heparin. ( H ) Immunofluorescence of human nasal tissue with anti-HS Ab and 4', 6-diamidino-2-phenylindole (DAPI). Scale bar, 200 µm. ( I ) Binding of WT or BA.1 RBD-Fc, anti-ACE2 Ab, or anti-HS Ab to cell lines. Data are mean ± SEM of three to four technical replicates. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison tests in panel F ; * P < 0.05, ** P < 0.01, and **** P < 0.0001; ns, not significant. Data are representative of two to three independent experiments.

Article Snippet: Mouse anti-human ACE2 monoclonal antibody (mAb) (AC384, Adipogen), mouse anti-HS mAb (F58-10E4, Amzbio), mouse anti-CS mAb (CS-56, Sigma-Aldrich), rat anti-Flag-tag mAb (L5, BioLegend), mouse anti-human CD138 (syndecan-1) mAb (MI15, BioLegend), mouse anti-human glypican-4 mAb (961609, R&D Systems), mouse anti-human CD46 mAb (J4.48, Beckman), mouse anti-human CD59 mAb (p282[H19], BioLegend), mouse anti-human HLA class I mAb (W6/32, Institute of Development, Aging and Cancer, University of Tohoku), mouse anti-human CD51 (integrin αV) mAb (L230, Enzo), Alexa Fluor 555 goat anti-mouse IgM (Heavy chain) Ab (Thermo Scientific), allophycocyanin (APC)-conjugated goat anti-human IgG, Fcγ fragment specific Ab, APC-conjugated goat anti-mouse IgG, Fcγ fragment specific Ab, APC-conjugated goat anti-mouse IgM, μ chain specific Ab, APC-conjugated donkey anti-rat IgG (H + L) Ab, and APC-conjugated streptavidin (Jackson) were used.

Techniques: Binding Assay, CRISPR, Library Screening, Immunofluorescence, Comparison

Journal: mBio

Article Title: Evolutionary dynamics of heparan sulfate utilization by SARS-CoV-2

doi: 10.1128/mbio.01303-25

Figure Lengend Snippet: Enhanced binding of RBD to cell surface HS by the mutations acquired by the Omicron variant. ( A ) Structure of BA.1 RBD (PDB: 7WBP) with mutation sites compared to WT (blue spheres) and ACE2-binding sites (receptor-binding motif; red). ( B ) Schematic of the binding assay using biotinylated HSPG or ACE2 with B3GAT3 KO HEK293T cells expressing Flag-tagged RBD fused to a transmembrane domain (Flag-RBD-TM) or Flag-tagged whole spike protein (Flag-spike). ( C and D ) The binding of biotinylated HSPG ( C ) or ACE2 ( D ) to Flag-RBD-TM transfectants of BA.1-based revertants is shown. Each revertant contains a single mutation reverted to the WT sequence. The expression levels of Flag-RBD-TM were adjusted by anti-Flag Ab staining. Data were normalized to the binding of BA.1. The dashed horizontal red lines indicate the value of parental BA.1. ( E and F ) The binding of biotinylated HSPG ( E ) or ACE2 ( F ) to Flag-spike transfectants of BA.1-based revertants is shown. The expression levels of Flag-spike were adjusted by anti-Flag Ab staining. Data are mean ± SEM of three to four biological replicates. Each dot represents one independent experiment. Statistical analysis was performed using unpaired two-tailed Student’s t -tests between parental BA.1 and each revertant; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Mouse anti-human ACE2 monoclonal antibody (mAb) (AC384, Adipogen), mouse anti-HS mAb (F58-10E4, Amzbio), mouse anti-CS mAb (CS-56, Sigma-Aldrich), rat anti-Flag-tag mAb (L5, BioLegend), mouse anti-human CD138 (syndecan-1) mAb (MI15, BioLegend), mouse anti-human glypican-4 mAb (961609, R&D Systems), mouse anti-human CD46 mAb (J4.48, Beckman), mouse anti-human CD59 mAb (p282[H19], BioLegend), mouse anti-human HLA class I mAb (W6/32, Institute of Development, Aging and Cancer, University of Tohoku), mouse anti-human CD51 (integrin αV) mAb (L230, Enzo), Alexa Fluor 555 goat anti-mouse IgM (Heavy chain) Ab (Thermo Scientific), allophycocyanin (APC)-conjugated goat anti-human IgG, Fcγ fragment specific Ab, APC-conjugated goat anti-mouse IgG, Fcγ fragment specific Ab, APC-conjugated goat anti-mouse IgM, μ chain specific Ab, APC-conjugated donkey anti-rat IgG (H + L) Ab, and APC-conjugated streptavidin (Jackson) were used.

Techniques: Binding Assay, Variant Assay, Mutagenesis, Expressing, Sequencing, Staining, Two Tailed Test

Journal: mBio

Article Title: Evolutionary dynamics of heparan sulfate utilization by SARS-CoV-2

doi: 10.1128/mbio.01303-25

Figure Lengend Snippet: Inhibition of HS binding to Omicron BA.1 spike by anti-RBD neutralizing Abs from BA.1-infected patients. ( A ) Binding of anti-RBD Abs from BA.1-infected patients to BA.1 spike. B3GAT3 KO HEK293T cells lacking HS were transfected with BA.1 spike and were used for Ab binding. ( B ) Inhibition of HSPG or ACE2 binding to BA.1 spike by anti-RBD Abs. HSPG or ACE2 binding to B3GAT3 KO HEK293T cells transfected with BA.1 spike was analyzed in the presence or absence of anti-RBD Abs. The maximum binding inhibition of Abs for HSPG or ACE2 binding to BA.1 spike is shown. Data are mean ± SEM of three technical replicates. Data are representative of two independent experiments.

Article Snippet: Mouse anti-human ACE2 monoclonal antibody (mAb) (AC384, Adipogen), mouse anti-HS mAb (F58-10E4, Amzbio), mouse anti-CS mAb (CS-56, Sigma-Aldrich), rat anti-Flag-tag mAb (L5, BioLegend), mouse anti-human CD138 (syndecan-1) mAb (MI15, BioLegend), mouse anti-human glypican-4 mAb (961609, R&D Systems), mouse anti-human CD46 mAb (J4.48, Beckman), mouse anti-human CD59 mAb (p282[H19], BioLegend), mouse anti-human HLA class I mAb (W6/32, Institute of Development, Aging and Cancer, University of Tohoku), mouse anti-human CD51 (integrin αV) mAb (L230, Enzo), Alexa Fluor 555 goat anti-mouse IgM (Heavy chain) Ab (Thermo Scientific), allophycocyanin (APC)-conjugated goat anti-human IgG, Fcγ fragment specific Ab, APC-conjugated goat anti-mouse IgG, Fcγ fragment specific Ab, APC-conjugated goat anti-mouse IgM, μ chain specific Ab, APC-conjugated donkey anti-rat IgG (H + L) Ab, and APC-conjugated streptavidin (Jackson) were used.

Techniques: Inhibition, Binding Assay, Infection, Transfection

Journal: mBio

Article Title: Evolutionary dynamics of heparan sulfate utilization by SARS-CoV-2

doi: 10.1128/mbio.01303-25

Figure Lengend Snippet: HS-dependent infection of the Omicron variants to low-level ACE2-expressing cells. ( A ) Infection of D614G or BA.1 pseudovirus to mock or ACE2-Tf HEK293T cells pretreated with (+) or without (–) heparinase. ( B ) Infection of D614G or BA.1 pseudovirus to HEK293T cells at different heparin concentrations. ( C ) Infection of authentic SARS-CoV-2 BA.1.18 variant to HEK293T cells at different heparin concentrations. Viral RNA in the supernatants at 24 hours post-inoculation (hpi) is shown. ( D ) Infection of authentic SARS-CoV-2 WT or BA.1.18 variant to primary human nasal epithelial cells in the presence (+) or absence (–) of 100 µg/mL heparin. Viral RNA in apical washes at 1 and 24 hpi or in cell lysates at 24 hpi is shown. Lysate RNA was normalized to Actb . ( E ) Titration of BA.2 or BA.4/5 pseudovirus using VSV-G-Tf HEK293T cells. ( F ) Infection of D614G, BA.1, BA.2, or BA.4/5 pseudovirus to ACE2-Tf HEK293T cells or to mock, B3GAT3 KO, or B3GAT3-Tf B3GAT3 KO HEK293T cells. ( G ) Infection of D614G, BA.1, BA.2, or BA.4/5 pseudovirus to mock, SLC35B2 KO, or SLC35B2-Tf SLC35B2 KO HEK293T cells. ( H ) Infection of authentic SARS-CoV-2 WT, BA.1.18 or BA.5 variant to mock, B3GAT3 KO, or B3GAT3-Tf B3GAT3 KO HEK293T cells. Viral RNA in supernatants or cell lysates at 24 hpi is shown. Lysate RNA was normalized to Actb . ( I ) Infection of D614G, BA.1, or BA.1-based revertant (A484E, R493Q, and R498Q) pseudovirus to HEK293T cells. Data are mean ± SEM of three to four technical replicates. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison tests in panels A and D , two-way ANOVA with Dunnett’s multiple comparison test compared to B3GAT3 KO HEK293T cells in panels F and H , two-way ANOVA with Dunnett’s multiple comparison test compared to SLC35B2 KO HEK293T cells in panel G , and unpaired two-tailed Student’s t -tests between parental BA.1 and each revertant in panel I ; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; ns, not significant. Data are representative of two to three independent experiments.

Article Snippet: Mouse anti-human ACE2 monoclonal antibody (mAb) (AC384, Adipogen), mouse anti-HS mAb (F58-10E4, Amzbio), mouse anti-CS mAb (CS-56, Sigma-Aldrich), rat anti-Flag-tag mAb (L5, BioLegend), mouse anti-human CD138 (syndecan-1) mAb (MI15, BioLegend), mouse anti-human glypican-4 mAb (961609, R&D Systems), mouse anti-human CD46 mAb (J4.48, Beckman), mouse anti-human CD59 mAb (p282[H19], BioLegend), mouse anti-human HLA class I mAb (W6/32, Institute of Development, Aging and Cancer, University of Tohoku), mouse anti-human CD51 (integrin αV) mAb (L230, Enzo), Alexa Fluor 555 goat anti-mouse IgM (Heavy chain) Ab (Thermo Scientific), allophycocyanin (APC)-conjugated goat anti-human IgG, Fcγ fragment specific Ab, APC-conjugated goat anti-mouse IgG, Fcγ fragment specific Ab, APC-conjugated goat anti-mouse IgM, μ chain specific Ab, APC-conjugated donkey anti-rat IgG (H + L) Ab, and APC-conjugated streptavidin (Jackson) were used.

Techniques: Infection, Expressing, Variant Assay, Titration, Comparison, Two Tailed Test

Journal: mBio

Article Title: Evolutionary dynamics of heparan sulfate utilization by SARS-CoV-2

doi: 10.1128/mbio.01303-25

Figure Lengend Snippet: TMPRSS2 cleaves cell surface HS proteoglycans. ( A ) Binding of WT or BA.1 RBD-Fc, anti-syndecan-1 (SDC1) Ab, anti-glypican-4 (GPC4) Ab, anti-HS Ab, anti-ACE2 Ab, anti-HLA class I Ab, anti-integrin αV (CD51) Ab, anti-CD46 Ab, or anti-CD59 Ab to HEK293T cells pretreated with (red line) or without (black line) trypsin. ( B ) Representative gating strategy for TMPRSS2-negative (GFP neg , black), low-expressing (GFP low , blue), or high-expressing (GFP high , red) cells in TMPRSS2/GFP-Tf HEK293T cells. ( C ) Top: representative histograms showing the binding of WT or BA.1 RBD-Fc, anti-SDC1 Ab, anti-GPC4 Ab, anti-HS Ab, anti-ACE2 Ab, anti-HLA class I Ab, anti-CD51 Ab, anti-CD46 Ab, or anti-CD59 Ab to TMPRSS2-negative (black line, GFP neg ), low-expressing (blue line, GFP low ) or high-expressing (red line, GFP high ) cells. Bottom: quantification of binding is shown as GMFI. ( D ) Infection of D614G or BA.1 pseudovirus to mock or ACE2-Tf HEK293T cells with (+) or without (–) TMPRSS2 expression. ( E ) Infection of authentic SARS-CoV-2 WT or BA.1.18 variant to mock or ACE2-Tf HEK293T cells with (+) or without (–) TMPRSS2 expression. Viral RNA in cell lysates at 24 hours post-inoculation is shown. Lysate RNA was normalized to Actb . Data are mean ± SEM of three to four technical replicates. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison tests in panel D and unpaired two-tailed Welch’s t -tests between WT and BA.1.18 in panel E ; * P < 0.05, ** P < 0.01, and **** P < 0.0001; ns, not significant. Data are representative of two to three independent experiments.

Article Snippet: Mouse anti-human ACE2 monoclonal antibody (mAb) (AC384, Adipogen), mouse anti-HS mAb (F58-10E4, Amzbio), mouse anti-CS mAb (CS-56, Sigma-Aldrich), rat anti-Flag-tag mAb (L5, BioLegend), mouse anti-human CD138 (syndecan-1) mAb (MI15, BioLegend), mouse anti-human glypican-4 mAb (961609, R&D Systems), mouse anti-human CD46 mAb (J4.48, Beckman), mouse anti-human CD59 mAb (p282[H19], BioLegend), mouse anti-human HLA class I mAb (W6/32, Institute of Development, Aging and Cancer, University of Tohoku), mouse anti-human CD51 (integrin αV) mAb (L230, Enzo), Alexa Fluor 555 goat anti-mouse IgM (Heavy chain) Ab (Thermo Scientific), allophycocyanin (APC)-conjugated goat anti-human IgG, Fcγ fragment specific Ab, APC-conjugated goat anti-mouse IgG, Fcγ fragment specific Ab, APC-conjugated goat anti-mouse IgM, μ chain specific Ab, APC-conjugated donkey anti-rat IgG (H + L) Ab, and APC-conjugated streptavidin (Jackson) were used.

Techniques: Binding Assay, Expressing, Infection, Variant Assay, Comparison, Two Tailed Test