gene fragment encoding rhccr5  (Addgene inc)

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Receptor binding and fusogenicity of S proteins of omicron variants (A) Amino Acid sequence alignment of RBDs from different omicron variants. Dashed lines represent residues identical to WT. Residues of RBM are shown in red. (B and C) Cleavage of S proteins from different omicron variants. Plasmids encoding codon-optimized S proteins were transfected into HEK293T cells and the cells were then lysed at 40 h post-transfection. The expression of S proteins was detected by western blot using rabbit polyclonal anti-S2 antibodies. Actin served as the loading control. Experiments were done three times and one representative was shown. Band densities were quantified using ImageLab software to determine the cleaved/FL ratio and normalized to WT. FL S, full-length spike protein. (D) Binding of soluble hACE2 to S proteins of omicron variants. HEK293T cells transiently expressing the indicated S proteins were incubated with biotinylated hACE2 followed by Alexa Fluor 488-conjugated streptavidin and then analyzed by flow cytometry. Experiments were done three times, and one representative was shown. (E and F) Cell-cell fusion mediated by S proteins of omicron variants. HEK293T cells were co-transfected with indicated S protein and eGFP. The cells were detached with trypsin at 48h post-transfection and overlaid on the HEK293 cells expressing hACE2 or Calu3 cells. After 2 h of incubation, five randomly selected images of syncytia were captured using a fluorescent microscope. Fusion areas were quantified using ImageJ software and normalized to WT as fusion efficiency. Experiments were done three times in triplicate and one representative is shown. The statistical difference relative to WT was determined by one-way ANOVA with Dunnett’s multiple testing correction ( n = 3 for (C) and (D); n = 5 for (E) and (F)). The statistical difference between BA.2.86 and JN.1 was determined by a two-tailed t-test ( n = 3 for (C) and (D); n = 5 for (E) and (F)). Error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Scale bar = 200μm. See also .

Article Snippet: The plasmids encoding animal ACE2 orthologs and hACE2 were synthesized by Sango Biotech (Shanghai, China) and cloned into the p3×Flag-CMV14 vector between the Hind III and BamH I sites.

Techniques: Binding Assay, Sequencing, Transfection, Expressing, Western Blot, Control, Software, Incubation, Flow Cytometry, Microscopy, Two Tailed Test

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Transduction of various omicron S pseudovirions on 293/hACE2 and Calu3 cells (A) S proteins incorporation in pseudovirions. Pseudovirions with different omicron S proteins were pelleted down by centrifugation through 20% sucrose cushion and separated in a 10% SDS-PAGE. Detection of S proteins in pseudovirions was performed by Western blot using rabbit polyclonal anti-S2 antibodies. The p24 served as the loading controls. Experiments were done three times and one representative was shown. (B and C) Entry of pseudovirions of omicron variants. HEK 293/hACE2 and Calu3 cells were transduced with omicron S pseudovirions and lysed at 40 h post-transduction. The transduction efficiencies were determined according to luciferase activities. Experiments were done three times in triplicate and one representative is shown. The statistical difference relative to WT was determined by one-way ANOVA with Dunnett’s multiple testing correction ( n = 3). The statistical difference between BA.2.86 and JN.1 was determined by a two-tailed t-test ( n = 3). Error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The plasmids encoding animal ACE2 orthologs and hACE2 were synthesized by Sango Biotech (Shanghai, China) and cloned into the p3×Flag-CMV14 vector between the Hind III and BamH I sites.

Techniques: Transduction, Centrifugation, SDS Page, Western Blot, Luciferase, Two Tailed Test

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Entry of different omicron pseudovirions on HEK293 cells expressing various animal ACE2s (A) Analysis of expression of various ACE2s on HEK293 cell surface by cell surface biotinylation assay. HEK293 cells transiently expressing different FLAG-tagged different ACE2s were labeled with EZ-link Sulfo-NHS-LC-LC-biotin on ice, and lysed with RIPA buffer. Biotinylated proteins were enriched with NeutraAvidin beads and detected by Western blot using mouse monoclonal anti-FLAG M2 antibody. Human Leukocyte Antigen (HLA) and actin served as controls. WCL, whole cell lysate. (B) Relative transduction efficiencies of different omicron variant pseudovirions on HEK 293 cells expressing various ACE2 orthologs. HEK293 cells transiently expressing different animal ACE2s were transduced with pseudovirions of WT, BA.1, XBB.1.16, EG.5.1, BA.2.86 and JN.1. Relative transduction efficiencies were normalized to WT/hACE2(100%). Experiments were performed three times in triplicate, and the averages are shown as the heatmap. (C) Statistic analyses of differences of transduction efficiencies between different omicron variant pseudovirion by Wilcoxon test. Significant differences indicated by p < 0.05 are highlighted in green background. See also .

Article Snippet: The plasmids encoding animal ACE2 orthologs and hACE2 were synthesized by Sango Biotech (Shanghai, China) and cloned into the p3×Flag-CMV14 vector between the Hind III and BamH I sites.

Techniques: Expressing, Cell Surface Biotinylation Assay, Labeling, Western Blot, Transduction, Variant Assay

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Receptor binding of omicron variants to various animal ACE2s (A) Relative receptor binding efficiencies of RBDs of WT, BA.1, XBB.1.16, EG.5.1, BA.2.86, and JN.1 variants to various animal ACE2s. HEK293T transiently expressing different animal ACE2s were detached with EDTA and incubated with mouse Fc-tagged RBDs of WT, BA.1, XBB.1.16, EG.5.1, BA.2.86, or JN.1, followed by FITC-conjugated polyclonal goat anti-mouse antibodies. The cells were analyzed by flow cytometry. Receptor binding efficiencies were calculated according to WT/hACE2, set as 100%. Experiments were performed three times, and the averages are shown in the heatmap. (B) Statistic analyses of levels of receptor binding between different omicron variant S protein by Wilcoxon test. Significant differences indicated by p < 0.05 are highlighted in green background.

Article Snippet: The plasmids encoding animal ACE2 orthologs and hACE2 were synthesized by Sango Biotech (Shanghai, China) and cloned into the p3×Flag-CMV14 vector between the Hind III and BamH I sites.

Techniques: Binding Assay, Expressing, Incubation, Flow Cytometry, Variant Assay

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Cell-cell fusion mediated by different omicron variant S proteins on HEK 293 cells expressing various animal ACE2s (A) Relative fusion efficiencies of WT, BA.1, XBB.1.16, EG.5.1, BA.2.86, and JN.1 on HEK 293 cells expressing animal ACE2 orthologs. HEK 293T cells transiently co-expressing S protein and eGFP were detached with trypsin and overlaid on HEK 293 cells expressing different animal ACE2s. After 2 h of incubation, the images of syncytia were captured using a fluorescent microscope. Fusion areas were quantified using ImageJ software and normalized to WT/hACE2, set as 100%. Experiments were performed three times, and the averages are shown in the heatmap. (B) Statistic analyses of levels of cell-cell fusion between different omicron variant S proteins by Wilcoxon test. Significant differences indicated by p < 0.05 are highlighted in green background.

Article Snippet: The plasmids encoding animal ACE2 orthologs and hACE2 were synthesized by Sango Biotech (Shanghai, China) and cloned into the p3×Flag-CMV14 vector between the Hind III and BamH I sites.

Techniques: Variant Assay, Expressing, Incubation, Microscopy, Software

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Effect of L455S mutation on transduction efficiency and fusogenicity of XBB.1.16 S protein (A) Relative transduction efficiencies of XBB.1.16 and XBB.1.16-L455S on HEK 293 cells expressing different animal ACE2s. HEK 293 cells transiently expressing different animal ACE2s were transduced by either XBB.1.16 WT S or L455S mutant S pseudovirions. The luciferase activities were measured at 40 h post transduction. Relative transduction efficiencies were calculated based on WT/hACE2(100%). Experiments were performed three times in triplicates and the representative one was shown. (B) Cell-cell fusion mediated by XBB.1.16 and XBB.1.16-L455S S proteins. HEK 293T cells transiently co-expressing S protein and eGFP were detached with trypsin and overlaid on HEK 293 cells expressing different animal ACE2s. After 2 h of incubation, the five randomly selected images of syncytia were captured using a fluorescent microscope. Fusion areas were quantified using ImageJ software and normalized to WT/hACE2, set as 100%. Experiments were performed twice and the representative one was shown. Statistical analyses were done by Wilcoxon test. See also .

Article Snippet: The plasmids encoding animal ACE2 orthologs and hACE2 were synthesized by Sango Biotech (Shanghai, China) and cloned into the p3×Flag-CMV14 vector between the Hind III and BamH I sites.

Techniques: Mutagenesis, Transduction, Expressing, Luciferase, Incubation, Microscopy, Software

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Effect of L455S mutation on thermal and proteolytic stability on XBB.1.16 and BA.2.86 (A and B) Thermal stability of JN.1 compared to its parental BA.2.86 (left panel) and XBB.1.16–455S to XBB.1.16 (right panel). Pseudovirus of XBB.1.16, XBB.1.16–455S, BA.2.86 and JN.1 were centrifuged through 20% sucrose to remove serum and resuspended in serum-free DMEM, followed by incubation either at 37°C for the indicated time (A) or at the indicated temperature for 2 h (B). The virus suspension was used to transduce 293/hACE2 cells to access their remaining transduction capability. Experiments were performed three times in triplicate, and the representative one was shown. The statistical difference was determined by a multiple t-test ( n = 3). Error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (C) Proteolytic stability of JN.1 compared to its parental BA.2.86 (left panel) and XBB.1.16–455S to XBB.1.16 (right panel). Pseudovirus of XBB.1.16, XBB.1.16–455S, BA.2.86 and JN.1 were resuspended in serum-free DMEM and incubated with soluble hACE2(20 μg) at 37°C for 30 min, followed by incubation with TPCK-treated trypsin. The mixture was immediately boiled with loading buffer containing DTT and separated in a 10% SDS-PAGE. Detection was carried out using rabbit polyclonal anti-SARS-CoV-2 S2 antibodies (1:3000). The numbers below S protein blot are the relative quantification of ratio of S2’/S2 using Image Lab (Bio-Rad). Experiments were performed at least three times, and the representative one was shown.

Article Snippet: The plasmids encoding animal ACE2 orthologs and hACE2 were synthesized by Sango Biotech (Shanghai, China) and cloned into the p3×Flag-CMV14 vector between the Hind III and BamH I sites.

Techniques: Mutagenesis, Incubation, Virus, Suspension, Transduction, SDS Page, Quantitative Proteomics

Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 1. SARS-CoV-2 3CLpro is released from infected cells by cell lysis and secreted by an unconventional protein-secretion mechanism (A) Immunoblots of 3CLpro precipitated by 33% trichloroacetic acid (TCA) from the conditioned media or in lysates from mock-infected or A549-ACE2 cells 48 hpi with SARS-CoV-2 (MOI 4, n = 5, N = 2). stds, 3CLpro standards. Densitometry of 3CLpro relative to 10 ng 3CLpro standard. (B) 3CLpro, b-tubulin, and GAPDH immunoblots and densitometry of 3CLpro in 33% TCA-precipitated conditioned media or cell lysate from mock-infected or SARS-CoV-2-infected A549-ACE2 cells 48 hpi with MOI 0.1 or 1.0. Dotted line, intracellular 3CLpro/tubulin ratio in MOI 1.0 cell lysate (n = 3/group). (C) Immunoblots and densitometry of 3CLpro in 33% TCA-precipitated conditioned media compared to matched lysates from SARS-CoV-2-infected Vero76 cells 48 hpi (MOI 1) or mock-infected cells (n = 3/group). 3CLpro ppt, 10 ng of 3CLpro precipitated from cell-naive media as a control; std, 10 ng of 3CLpro standard. (D) Immunoblots of 3CLpro in conditioned media concentrated by ultrafiltration and cell lysates from HEK293 cells transfected with 3CLpro expression plasmid for 48 h (n = 8, N = 2). (E) LDH activity in conditioned medium collected from 3CLpro transfected HEK293 cells (48 h) compared with the maximum amount of LDH released from lysed HEK293 cells (n = 6, N = 2). Absorbance was measured at 490 nm with 680 nm correction. (F) Immunoblots and densitometry of 3CLpro in concentrated conditioned medium and cell lysates from HEK293 cells transiently transfected with plasmids encoding 3CLpro or catalytic inactive 3CLpro Cys145Ala for 48 h (n = 5, N = 2). (G) Immunoblots and densitometry of 3CLpro or IFN-l1 after 24 h culture ± Golgi inhibitor 2 mM monensin (n = 6, N = 2). (H) 3CLpro, GSDMD, and b-actin loading control immunoblots and GSDMD densitometry of lysates from mock-infected or SARS-CoV-2-infected A549-ACE2 cells 48 hpi, MOI 0.1 or 1.0 (n = 3/group). (I) Schematic of candidate mechanisms of 3CLpro release from SARS-CoV-2-infected cells. Antibodies: anti-3CLpro (1:1,000, in-house), anti-b-tubulin (1:2,000, clone: BT7R), anti-b-actin (1:2,000, ab8226), anti-GAPDH (1:2,000, clone: GA1R), anti- GSDMD N-terminal antibody (1:500, clone: W18029B), and anti-FLAG M2 (IFN-l1 in G) (1:1,000, F3165). Data are shown as mean ± SD; statistical analysis of two groups was performed by two-tailed unpaired t test, three or more groups by one-way ANOVA with Tukey’s post hoc test, with a significance threshold of p < 0.05.

Article Snippet: Lentiviruses were produced by co-transfection of HEK293T/ 17 cells with a transfer plasmid encoding ACE2 (Addgene #154981), the lentiviral packaging plasmid psPAX2 (Addgene #12260), and an envelope plasmid encoding VSV-G (Addgene #8454).

Techniques: Infection, Lysis, Western Blot, Control, Transfection, Expressing, Plasmid Preparation, Activity Assay, Two Tailed Test