Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42 reduces cholesterol abundance in the plasma membrane in general and lipid rafts in particular, and decreases lipid raft area more efficiently than atorvastatin (ATO). Control HEK293T (A) and Calu-3 (B) cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were labeled with cholesterol-binding mCherry-conjugated D4H*, the D434S mutant of domain 4 (D4) of C. perfringens theta-toxin. Fluorescence intensities correlating with plasma membrane cholesterol levels of at least 10,000 individual cells per sample were subsequently measured using a flow cytometer. The average intensity values obtained in n = 9 independent biological replicates, and their average values (± SEM) are plotted in the figure. (C) To examine changes in the cholesterol content of raft and non-raft microdomains of the plasma membrane, control HEK/ACE2 + TMPRSS2 cells and those treated as above were labeled with Alexa Fluor 647-conjugated cholera toxin subunit B (CTX-AF647), a lipid raft marker, and F66. F66 is a fluorescent indicator with spectral properties depending on the cholesterol-dependent local molecular order (dipole potential) of the membrane; therefore, this dye, combined with CTX-AF647, can provide information about the extent of cholesterol reduction separately in raft and non-raft membrane regions. Representative confocal microscopic images taken from the flat, bottom membrane region adjacent to the coverglass show F66 intensities detected in 2 wavelength ranges of emission (“F66 N*” and “F66 T*”), their ratio (“F66 emission ratio” calculated as T*/N* pixel by pixel), and CTX-AF647 intensities. Cell “membrane masks” selected manually in CTX images were segmented using the maxentropy algorithm to CTX-high “rafts” and CTX-low “non-rafts” corresponding to high- and low-intensity regions, respectively, as shown by the representative images. Violin plots were generated from median F66 emission ratio values determined separately for the CTX-high “raft” (D) and CTX-low “non-raft” (E) masks of n = 54 to 73 individual cells, which also display median values with quartiles. (F) Pixelwise distributions of the F66 emission ratio in CTX-high “rafts” and CTX-low “non-rafts” of control cells are displayed. For the quantification of the relative area of lipid rafts, as an alternative definition for raft regions, a threshold value of the F66 emission ratio was determined (green dashed line) and membrane pixels were considered as “F66 raft” and “F66 non-raft” regions when being above and below the threshold, respectively. (G) Violin plots were generated from the relative fraction of F66 raft pixels (“F66 raft area”) of individual cells, which also display median values with quartiles. (H) Representative images show changes in the lateral distribution of the F66 emission ratio on a color-scale image and reduction in the relative F66 raft area induced by 1 μM SH42. Throughout the figure, asterisks indicate significant differences compared to control samples (* P < 0.05, **** P < 0.0001), while hashes show that between samples treated with ATO and SH42 at identical concentrations ( # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Clinical Proteomics, Membrane, Control, Labeling, Binding Assay, Mutagenesis, Fluorescence, Flow Cytometry, Marker, Generated

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42 decreases ACE2 binding of SARS-CoV-2 spike receptor-binding domains (RBDs) more efficiently than ATO. (A) ACE2-expressing HEK/ACE2 + TMPRSS2 and Calu-3 control cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were incubated with the GFP-conjugated RBDs of the Wuhan-Hu-1 strain (WT), Delta, and Omicron BA.1 variants for 4 min. RBDs were applied at 0.2 and 1.0 μg/ml for HEK/ACE2 + TMPRSS2 and Calu-3 cells, respectively. Fluorescence intensities of at least 10,000 individual cells per sample were subsequently measured using a flow cytometer. (B) Representative RBD-GFP versus forward-scattered light intensity (FSC) density plots demonstrate decreases in the bound WT RBD-GFP in response to 1 μM SH42 in HEK/ACE2 + TMPRSS2 cells. Dashed lines represent average values of the fluorescence intensity obtained in the displayed representative samples. The average intensities obtained in n = 9 independent biological replicates and normalized to the mean value determined in untreated control samples, and their average values (± SEM) are plotted for WT, Delta, and Omicron BA.1 variants in HEK/ACE2 + TMPRSS2 (C) and Calu-3 (D) cells. Throughout the figure, asterisks indicate significant differences compared to control samples (* P < 0.05, ** P < 0.01, **** P < 0.0001), while hashes show those between samples treated with ATO and SH42 at identical concentrations ( ### P < 0.001, #### P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Binding Assay, Expressing, Control, Incubation, Fluorescence, Flow Cytometry

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42-induced reduction in ACE2 binding of WT SARS-CoV-2 spike RBDs negatively correlates with the applied RBD concentration. ACE2-expressing HEK/ACE2 + TMPRSS2 (A) and Calu-3 (B) control cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were incubated with different concentrations of the GFP-conjugated RBDs of the Wuhan-Hu-1 strain (WT) for 4 min. Fluorescence intensities of at least 10,000 individual cells per sample were subsequently measured using a flow cytometer. The extents of inhibition (calculated as 1 − average of treated/average of control) were determined in n = 9 independent biological replicates, and their average values (± SEM) are plotted as a function of the applied RBD concentration ranging between 0.1 and 5 μg/ml for HEK/ACE2 + TMPRSS2 and between 1 and 10 μg/ml for Calu-3 cells. Asterisks indicate significant differences between samples treated with the lowest versus highest RBD concentrations for each treatment (** P < 0.01, **** P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Binding Assay, Concentration Assay, Expressing, Control, Incubation, Fluorescence, Flow Cytometry, Inhibition

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42 inhibits the cellular entry of SARS-CoV-2 spike trimers more efficiently than ATO. Control HEK/ACE2 + TMPRSS2 and Calu-3 cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were incubated for 4 h in the presence of WT, Delta, or Omicron BA.1 SARS-CoV-2 spike trimers conjugated with Alexa Fluor 488 (AF488-trimers) and labeled with F66. (A) Representative orthogonal views of confocal Z-stack images of F66 for the visualization of the plasma membrane and AF488-trimers to estimate entry demonstrate notable trimer accumulation in the intracellular space of untreated control HEK/ACE2 + TMPRSS2 cells. During image analysis, pixels corresponding to plasma membrane and intracellular pixels were segmented based on F66 Z-stack images. Markers were manually placed inside cells (green circles in the grayscale orthogonal view), and a MATLAB implementation of the 3D watershed algorithm identified the intracellular space of cells and their membrane (colored regions and red lines in the orthogonal view in the middle, respectively, and their overlay image displayed on the right). (B) Representative 3D reconstruction images displaying AF488 fluorescence intensities on a green-red color scale above a threshold intensity overlaid on intracellular pixels of individual cells (in transparent blue) demonstrate decreases in the amount of intracellular WT trimers in response to 1 μM SH42. Subsequently, the average fluorescence intensity values emitted by AF488-trimers were calculated exclusively from data of intracellular pixels for individual cells. The average intensities obtained in n = 400 to 600 HEK/ACE2 + TMPRSS2 (C) and Calu-3 (D) cells and normalized to the median value determined in untreated control samples are plotted along with median values with quartiles for WT, Delta, and Omicron BA.1 trimer variants. Asterisks indicate significant differences compared to control samples (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001), while hashes show that between samples treated with ATO and SH42 at identical concentrations ( # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA. Lognormal functions fitted to normalized mean intracellular AF488-trimer fluorescence intensity histograms of individual HEK/ACE2 + TMPRSS2 (E) and Calu-3 (F) cells also demonstrate the effects of ATO and SH42 on the internalization of WT, Delta, and Omicron BA.1 trimer variants.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Control, Incubation, Labeling, Clinical Proteomics, Membrane, Fluorescence

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: SH42 decreases cell surface ACE2 expression and its colocalization with lipid rafts more efficiently than ATO. (A) Control HEK/ACE2 + TMPRSS2 and Calu-3 cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were labeled with Alexa Fluor 488-conjugated anti-ACE2 antibodies (AF488-anti-ACE2). Fluorescence intensities of at least 10,000 individual cells per sample were subsequently measured using a flow cytometer. The average intensities obtained in n = 10 independent biological replicates and normalized to the mean value determined in untreated control samples, and their average values (± SEM) are plotted in the panel. (B) Control cells and those treated as above were labeled with AF488-anti-ACE2 and Alexa Fluor 647-conjugated cholera toxin subunit B (AF647-CTX). Representative confocal microscopic images taken from the flat, bottom membrane region adjacent to the coverglass show AF488-anti-ACE2 and AF647-CTX intensities, and their overlay, while the colocalization of the 2 signals and its changes in response to 1 μM SH42 are displayed in representative dot plots obtained from pixelwise fluorescence intensities. (C) Violin plots were generated from Pearson correlation coefficient values between fluorescence intensities of the 2 applied fluorophores determined from pixelwise data of n = 81 to 90 individual cells, which also display median values with quartiles. Throughout the figure, asterisks indicate significant differences compared to control samples (* P < 0.05, ** P < 0.01, **** P < 0.0001), while hashes show that between samples treated with ATO and SH42 at identical concentrations ( ### P < 0.001, #### P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Expressing, Control, Labeling, Fluorescence, Flow Cytometry, Membrane, Generated

Journal: Research

Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin

doi: 10.34133/research.1280

Figure Lengend Snippet: DHCR24 inhibition by its selective blocker SH42 is a potential novel therapeutic approach to suppress initial membrane-coupled events of SARS-CoV-2 infection. SH42, a novel steroidal highly selective and potent DHCR24 inhibitor, interferes with ACE2 binding of SARS-CoV-2 spike RBDs and cellular uptake of spike proteins. By efficiently decreasing cholesterol levels of the host cell plasma membrane and causing the concomitant disruption of lipid raft microdomains, SH42 decreases cell surface levels of ACE2 and, in addition, reduces raft partitioning of the receptor protein, thereby altering its local microenvironment required for an efficient ACE2-mediated cellular binding and uptake of the virus. As a result, early membrane-coupled events of SARS-CoV-2 infection are inhibited as mirrored by the decreased binding of spike RBDs to host membrane and decreased cellular uptake of spike trimers, and culminate in decreased cellular infection with replication-competent SARS-CoV-2 virions.

Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).

Techniques: Inhibition, Membrane, Infection, Binding Assay, Clinical Proteomics, Disruption, Virus

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Generation of the SARS-CoV-2 N conditional knock-in mouse model. a Schematic diagrams illustrating the knock-in strategy, in which the CAG-loxP-Stop-loxP-SARS-CoV-2 N-WPRE-PolyA sequence was inserted into the Tigre locus on chromosome 9, using the K18-hACE2 KI mouse background (N-hACE2 mouse). The stop sequence was flanked by two loxP sites, and upon expression of Cre recombinase, the stop sequence located between these loxP sites was excised. N-hACE2 mice were crossed with Rosa26 SA-CreERT2 or Sftpc-IRES-iCre mice, resulting in the generation of SA-N-hACE2 (TAM-inducible systemic Cre expression) and Sftpc-N-hACE2 (lung-specific Cre expression) mice. b N-hACE2 mouse genotyping by PCR showed the presence of N gene and hACE2 gene fragments. M: GimiRun DM5000 DNA Marker. c PCR confirmed excision of the stop sequence between loxP sites in SA-N-hACE2 mice after 5 days of TAM injection. Western blot analysis of SARS-CoV-2 N protein expression in multiple tissues from SA-N-hACE2 mice after TAM induction ( d ) and from Sftpc-N-hACE2 mice ( e ). f Immunofluorescence staining of lung sections was conducted using 4,6-diamidino-2-phenylindole (DAPI, blue), an anti-SARS-CoV-2 N antibody (red), and an anti-hACE2 antibody (green) to evaluate the expression of SARS-CoV-2 N and hACE2 in lung cells

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Knock-In, Sequencing, Expressing, Marker, Injection, Western Blot, Immunofluorescence, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: SA-N-hACE2 mice are susceptible to SARS-CoV-2 ΔN/GFP-HiBiT infection. a Experimental design for intranasal infection. After TAM treatment, SA-N-hACE2 mice were infected either with 5 × 10 4 or 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. Tissue samples were collected at the indicated dpi. b Changes in the weights of the mice are shown ( n = 4 per group). c qRT‒PCR was used to quantify viral loads in tissues at 7 dpi ( n = 4 per group). The viral loads ( d ) and luminescence ( e ) were measured in the lungs collected at 2, 4, and 7 dpi ( n = 4 per group). f Immunofluorescence staining of lung sections at 2, 4, and 7 dpi was conducted using DAPI (blue) and an anti-GFP antibody (green) specific to GFP in SARS-CoV-2 ΔN/GFP-HiBiT. g – j Pathological changes observed using H&E staining in lung ( g ) and brain ( i ) tissues from SA-N-hACE2 mice challenged with 1 × 10 6 TCID 50 at 0, 2, 4, and 7 dpi. Pathology scores for the lungs ( h ) and brain ( j ) were calculated ( n = 4 per group). Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( h ). ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ND not detected

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Immunofluorescence, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Sftpc-N-hACE2 mice are susceptible to SARS-CoV-2 ΔN/GFP-HiBiT infection. a Illustration of the protocol for intranasal infection with tissue samples collected at the indicated dpi. Sftpc-N-hACE2 mice were infected with 5 × 10 4 or 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. b Changes in the body weights of the mice are shown ( n = 4 per group). c Viral loads in the tissues obtained at 7 dpi were quantified via qRT‒PCR ( n = 4 per group). The viral loads ( d ) and luminescence ( e ) were measured in the lungs collected at 2, 4, and 7 dpi ( n = 4 per group). f Immunofluorescence staining of lung sections at 2, 4, and 7 dpi was conducted using DAPI (blue) and an anti-GFP antibody (green) specific to GFP in SARS-CoV-2 ΔN/GFP-HiBiT. g – j Pathological changes in the lungs ( g ) and brains ( i ) of Sftpc-N-hACE2 mice challenged with 1 × 10 6 TCID 50 at 0, 2, 4, and 7 dpi were assessed using H&E staining. Pathology scores for the lungs ( h ) and brains ( j ) are shown ( n = 4 per group). Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( h ). ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ND not detected

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Immunofluorescence, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: SARS-CoV-2 ΔN/GFP-HiBiT infection in Sftpc-N-hACE2 mice can lead to lethal disease. a The schematic outlines the procedure for infecting the mice with 5 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. At 0, 7, 10, 14, and 21 dpi, four mice were euthanized at each time point for sample collection. Mice that experienced a loss of more than 20% of their initial body weight were euthanized as a humane endpoint. Mice were monitored for body weight changes ( b ) and survival ( c ) ( n = 16). E gene copies in lung ( d ) and brain ( e ) tissues were quantified via qRT‒PCR. f – i Pathological changes were assessed using H&E staining in the lungs ( f ) and brains ( g ). Pathology scores for the lungs ( h ) and brains ( i ) were recorded ( n = 4 per group). j Immunofluorescence staining of brain sections was performed using DAPI (blue) and an anti-GFP antibody (green) specific to GFP in SARS-CoV-2 ΔN/GFP-HiBiT. k Immunofluorescence analysis was performed on brain sections from mock-infected mice, K18-hACE2 KI mice challenged with live SARS-CoV-2, and both SA-N-hACE2 and Sftpc-N-hACE2 mice exposed to SARS-CoV-2 ΔN/GFP-HiBiT. The sections were stained with anti-IBA1 (red) and anti-CD68 (green) antibodies. l Light-sheet imaging of cleared lung tissues from Sftpc-N-hACE2 mice at 7 dpi confirmed the distribution of SARS-CoV-2 N in lung epithelial cells and SARS-CoV-2 ΔN/GFP-HiBiT infection. Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( h ). ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ND not detected

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Staining, Immunofluorescence, Imaging

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Transcriptomic profiling of the lungs of SA-N-hACE2 and Sftpc-N-hACE2 mice revealed distinct molecular features. a Scheme illustrating the transcriptome sequencing. Gene expression heatmaps of IFN-I-related genes ( b ) and cytokines and chemokines ( c ) in the lungs of the mice. Mock: PBS-treated mice; the SA-N-hACE2 and Sftpc-N-hACE2 mice were infected with 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. d Bubble plot showing gene set enrichment analysis (GSEA) results for pathways enriched in the lungs of the infected mice. The color scale indicates normalized enrichment scores (NES), while the size of the bubbles corresponds to −log 10 ( p values)

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Sequencing, Gene Expression, Infection

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Assessment of anti-SARS-CoV-2 therapeutics using SA-N-hACE2 and Sftpc-N-hACE2 mouse models. a This schematic diagram illustrates the procedure for evaluating the neutralizing antibody 7B3 and NMV. SA-N-hACE2, Sftpc-N-hACE2, and K18-hACE2 KI mice received 10 mg/kg of 7B3 at 1 dpi or 300 mg/kg NMV via oral gavage from 0–3 dpi. SA-N-hACE2 and Sftpc-N-hACE2 mice were challenged with 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT, whereas K18-hACE2 KI mice were infected with 3.57 × 10 2 TCID 50 of SARS-CoV-2 WT at 0 dpi. At 4 dpi, all mice were sacrificed, and lung samples were collected. The expression of the viral E gene in lung tissues from mock-, NMV- and 7B3-treated SA-N-hACE2 ( b ), Sftpc-N-hACE2 ( c ) and K18-hACE2 KI mice ( d ) was quantified via qRT‒PCR ( n = 4 per group). e – j Lung tissues from mock-, NMV-, and 7B3-treated SA-N-hACE2 ( e ), Sftpc-N-hACE2 ( f ), and K18-hACE2 KI ( g ) mice were analyzed using H&E staining to assess pathological changes, with histological scores documented for each group ( n = 4 per group) ( h – j ). Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( b – d , h – j ). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Expressing, Staining

Journal: EBioMedicine

Article Title: Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2.

doi: 10.1016/j.ebiom.2024.105281

Figure Lengend Snippet: Fig. 4: BTN3A2 competed with ACE2 to bind to the Spike protein. a: BTN3A2 isoform (BTN3A2-L-His or BTN3A2-S-His) interacted with ACE2. b: ACE2 interacted with BTN3A2-L. c: Endogenous BTN3A2 interacted with the ACE2. Procedures were similar to Fig. 3e d: Biolayer interferometry analysis of BTN3A2-L binding to immobilized ACE2-Fc. Different concentrations of BTN3A2-L (2000, 1000, 250, and 62.5 nM; corresponding to kinetic curves from top to bottom) were used. e: Biolayer interferometry analysis of BTN3A2-S binding to immobilized ACE2-Fc. Different concentrations of BTN3A2-S (1000 and 500 nM; corresponding to kinetic curves from top to bottom) were used. f-g: Competition ELISA analysis of competitive binding of BTN3A2-L with Spike RBD. f: Schematic of competition assays. g: Competition between ACE2 (ACE2-His-tagged) and BTN3A2-L (BTN3A2) for immobilized Spike S1 subunit (S1-Fc). ACE2 and S1-Fc protein or human IgG1-Fc and S1-Fc protein were incubated for 12 h, followed by the addition of dilution series of BTN3A2-L-His. Amount of S1 protein remaining in the presence of the competitor was determined by antihuman HRP through a colorimetric readout. h: BTN3A2-L competed with ACE2 to bind to the Spike S1. Procedures for IP and IB in (a-b) and (h) were similar to those in Fig. 3c.

Article Snippet: The membranes were then blocked with 5% non-fat dry milk in Tris-buffered saline (#9997, Cell Signaling Technology, USA) with 0.1% Tween 20 (P1379, Sigma, USA) (TBST) at room temperature for 2 h. Membranes were incubated with primary antibodies against His (1:5000, M20001M, Abmart, China), HA (1:5000, M20003M, Abmart, China), Flag (1:5000, M20008M, Abmart, China), ACE2 (1:1000, 66699-1-Ig, Proteintech, China), BTN3A2 (1:1000, TA500730, Origene, USA), Spike (1:1000, 28869-1-AP, Proteintech, www.thelancet.com Vol 107 September, 2024 China), and Tubulin (1:10,000, OTI3H2, ZSGB-Bio, China) overnight at 4 ◦C, respectively.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: EBioMedicine

Article Title: Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2.

doi: 10.1016/j.ebiom.2024.105281

Figure Lengend Snippet: Fig. 5: BTN3A2 inhibited ACE2 expression. a: Differential expression of BTN3A2 and ACE2 in scRNA-seq dataset of lung tissues from COVID-19 patients and uninfected individuals. Original dataset was reported in Melms et al.43 Significance was assessed by two-sided Wilcox rank sum test. b: Stable expression of BTN3A2 decreased ACE2 mRNA level in Huh7 cells. RNA was extracted from Huh7 cells overexpressing BTN3A2-L and BTN3A2-S, or control (Vector Huh7) cells (each group 1 × 105 cells). BTN3A2 and ACE2 mRNA levels were measured by qRT-PCR, with normalization to GAPDH. c-d: Overexpression of BTN3A2-L (c) and BTN3A2-S (d) suppressed ACE2 protein expression in HEK293T cells. Cells (5 × 105) were transfected with indicated expression vector or empty vector (each 1.25 μg) for 48 h, then harvested for western blotting. e: Dose-dependent inhibitory effect of BTN3A2-L on ACE2 protein expression. HEK293T cells (5 × 105) were transfected with indicated expression

Article Snippet: The membranes were then blocked with 5% non-fat dry milk in Tris-buffered saline (#9997, Cell Signaling Technology, USA) with 0.1% Tween 20 (P1379, Sigma, USA) (TBST) at room temperature for 2 h. Membranes were incubated with primary antibodies against His (1:5000, M20001M, Abmart, China), HA (1:5000, M20003M, Abmart, China), Flag (1:5000, M20008M, Abmart, China), ACE2 (1:1000, 66699-1-Ig, Proteintech, China), BTN3A2 (1:1000, TA500730, Origene, USA), Spike (1:1000, 28869-1-AP, Proteintech, www.thelancet.com Vol 107 September, 2024 China), and Tubulin (1:10,000, OTI3H2, ZSGB-Bio, China) overnight at 4 ◦C, respectively.

Techniques: Expressing, Quantitative Proteomics, Control, Plasmid Preparation, Quantitative RT-PCR, Over Expression, Transfection, Western Blot

Journal: EBioMedicine

Article Title: Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2.

doi: 10.1016/j.ebiom.2024.105281

Figure Lengend Snippet: Fig. 6: BTN3A2 suppressed ACE-mediated SARS-CoV-2 entry. a: Schematic of animal experiments with SARS-CoV-2 inoculation. BTN3A2-tg (n = 6) and WT mice (C57BL/6J, n = 7) were intranasally infected with AAV9-hACE2 (2 × 1012 viral genome copies) for 14 days, with all animals then infected with BA.2 (5 × 104 TCID50). All mice were euthanized at 3 dpi for tissue collection. b: Viral RNA copies in lung tissues of SARS- CoV-2-infected mice at 3 dpi. Each dot indicates log copies of viral genomic N gene, E gene, and subgenomic E (sgE) of a mixed sample of five lung lobes from an individual mouse. Viral copies are presented as mean ± SD. ***, P < 0.001; ANOVA with Dunnett’s multiple comparisons. c: Representative image of immunohistochemical staining of SARS-CoV-2 N protein in lung tissues of WT and BTN3A2-tg mice at 3 dpi. Scale bars: 500 μm for entire lung lobe section (left) and 100 μm for enlarged view of boxed areas labeled by numbers in entire section (right). d: Representative of image of H&E staining of lung sections in SARS-CoV-2-infected WT and BTN3A2-tg mice. e: AAV-mediated hACE2 expression in lungs tissues of WT (C57BL/6J, n = 7) and BTN3A2-tg mice (n = 6) at 3 dpi. f: Representative image of immunohistochemical staining for ACE2 and BTN3A2 in lung tissues of WT and BTN3A2-tg mice at 3 dpi. Scale bars were same as those in (c).

Article Snippet: The membranes were then blocked with 5% non-fat dry milk in Tris-buffered saline (#9997, Cell Signaling Technology, USA) with 0.1% Tween 20 (P1379, Sigma, USA) (TBST) at room temperature for 2 h. Membranes were incubated with primary antibodies against His (1:5000, M20001M, Abmart, China), HA (1:5000, M20003M, Abmart, China), Flag (1:5000, M20008M, Abmart, China), ACE2 (1:1000, 66699-1-Ig, Proteintech, China), BTN3A2 (1:1000, TA500730, Origene, USA), Spike (1:1000, 28869-1-AP, Proteintech, www.thelancet.com Vol 107 September, 2024 China), and Tubulin (1:10,000, OTI3H2, ZSGB-Bio, China) overnight at 4 ◦C, respectively.

Techniques: Infection, Immunohistochemical staining, Staining, Labeling, Expressing

Journal: Cardiovascular Research

Article Title: Blood flow patterns regulate PCSK9 secretion via MyD88-mediated pro-inflammatory cytokines

doi: 10.1093/cvr/cvz262

Figure Lengend Snippet: TLR4 signalling regulates distribution of PCSK9 expression along the aorta. (A) Effect of TLR4 inhibitor TAK-242, MyD88 inhibitor Pepinh-MYD, TRIF inhibitor Pepinh-TRIF, and NF-κB Helenalin on PCSK9 levels, measured by ELISA. (B) LPS injection induces expression of pro-inflammatory cytokines IL-1β, IL-18, MCP-1, IL-6, TNFα, IL-12, IFNγ, and GM-CSF. Measured by ELISA in serum on day 3 HFD group; rabbits treated with or without LPS. Bar graphs represent data compiled from three independent experiments (n = 7 rabbit per genotype), shown as mean ± standard deviation. The significances between two groups were tested by unpaired t-test; Multiple comparisons were analysed by one-way ANOVA, followed by Tukey’s post hoc comparisons test (**P < 0.01; ****P < 0.0001).

Article Snippet: Secretion of PCSK9, IL-1β, MCP-1, IL-6, TNFα, IL-12, and IFNγ was measured in rabbit sera or aorta by using a rabbit enzyme-linked immunosorbent assay (ELISA) kit for PCSK9, IL-1β, IL-18, MCP-1, IL-6, TNFα, IL-12, IFNγ, and GM-CSF (MyBioSource, Inc., San Diego, CA, USA); Rabbit ELISA kit for IL-18 and GM-CSF were from LSBio (Seattle, WA, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Injection, Standard Deviation

Journal: Frontiers in Plant Science

Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

doi: 10.3389/fpls.2020.604663

Figure Lengend Snippet: Schematic representation of plant expression vector pBYR2e-ACE2-Fc used in the present study (A) . Diagrammatic representation showing the binding of plant-produced ACE2-Fc with SARS-CoV-2 thereby preventing the virus entry into the host cell (B) .

Article Snippet: After blocking, a 1:2,000 dilution of rabbit anti-ACE2 antibody (SinoBiological, United States) in 1xPBS was added into the wells and incubated for 2 h at 37°C.

Techniques: Expressing, Plasmid Preparation, Binding Assay, Produced

Journal: Frontiers in Plant Science

Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

doi: 10.3389/fpls.2020.604663

Figure Lengend Snippet: Expression profiles of ACE2-Fc in N. benthamiana leaves on days 2, 4, 6, 8, and 10 after agroinfiltration. Leaf necrosis (A) Quantification of plant-produced ACE2-Fc (B) . The infiltrated leaves were collected from 3 individual plants in each day post infiltration. Data were analyzed by indirect ELISA assay using ACE2-specific antibody and presented as mean ± SD of triplicates.

Article Snippet: After blocking, a 1:2,000 dilution of rabbit anti-ACE2 antibody (SinoBiological, United States) in 1xPBS was added into the wells and incubated for 2 h at 37°C.

Techniques: Expressing, Produced, Indirect ELISA

Journal: Frontiers in Plant Science

Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

doi: 10.3389/fpls.2020.604663

Figure Lengend Snippet: Analysis of purified plant-produced ACE2-Fc (lane 1) and commercial HEK293-produced ACE2-Fc (lane 2). Coomassie-stained SDS-PAGE under reducing (A) and non-reducing conditions (B) . Western blotting analysis under reducing condition with detection using a rabbit anti-ACE2 antibody (C) and an anti-human gamma-HRP conjugated antibody (D) . Western blotting analysis under non-reducing condition probed with a rabbit anti-ACE2 antibody (E) and an anti-human gamma-HRP conjugated antibody (F) .

Article Snippet: After blocking, a 1:2,000 dilution of rabbit anti-ACE2 antibody (SinoBiological, United States) in 1xPBS was added into the wells and incubated for 2 h at 37°C.

Techniques: Purification, Produced, Staining, SDS Page, Western Blot

Journal: Frontiers in Plant Science

Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

doi: 10.3389/fpls.2020.604663

Figure Lengend Snippet: Binding activity of the plant-produced ACE2-Fc with the commercial receptor binding domain of SARS-CoV-2 (SARS-CoV-2 RBD) from Sf9 cells was analyzed by ELISA. PBS buffer and S1 protein of PEDV were used as negative controls. Data are presented as mean ± SD of triplicates.

Article Snippet: After blocking, a 1:2,000 dilution of rabbit anti-ACE2 antibody (SinoBiological, United States) in 1xPBS was added into the wells and incubated for 2 h at 37°C.

Techniques: Binding Assay, Activity Assay, Produced, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Plant Science

Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

doi: 10.3389/fpls.2020.604663

Figure Lengend Snippet: Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the pre-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.

Article Snippet: After blocking, a 1:2,000 dilution of rabbit anti-ACE2 antibody (SinoBiological, United States) in 1xPBS was added into the wells and incubated for 2 h at 37°C.

Techniques: Produced, Inhibition, Neutralization, Infection

Journal: Frontiers in Plant Science

Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

doi: 10.3389/fpls.2020.604663

Figure Lengend Snippet: Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the post-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.

Article Snippet: After blocking, a 1:2,000 dilution of rabbit anti-ACE2 antibody (SinoBiological, United States) in 1xPBS was added into the wells and incubated for 2 h at 37°C.

Techniques: Produced, Inhibition, Neutralization, Infection