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