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: Mouse polyclonal against FLAG-tag antibodies , SinoBiological Inc (Beijing, China) , Cat#109143-MM13.

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: Mouse polyclonal against FLAG-tag antibodies , SinoBiological Inc (Beijing, China) , Cat#109143-MM13.

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: 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: Mouse polyclonal against FLAG-tag antibodies , SinoBiological Inc (Beijing, China) , Cat#109143-MM13.

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: 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: Mouse polyclonal against FLAG-tag antibodies , SinoBiological Inc (Beijing, China) , Cat#109143-MM13.

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

Journal: bioRxiv

Article Title: Coevolving Residues Distant from the Ligand Binding Site are Involved in GAF Domain Function

doi: 10.1101/2024.08.07.605472

Figure Lengend Snippet: (A) Schematic showing the mechanism of the BRET 2 -based, full-length PDE5A2 conformational biosensor in the absence and presence of cGMP. (B) Western blot analysis of the cell lysates prepared from HEK293T cells transfected with either the WT or mutant full-length PDE5A2 biosensor plasmid constructs probed using an anti-GFP antibody showing the expression of biosensor constructs. (C) Graphs showing bioluminescence spectra obtained from lysates prepared from cells expressing the WT and mutant L267A and F295A mutant PDE5 biosensor constructs. Note the reduction in the GFP 2 emission peaks in the L267A and F295A mutant PDE5 biosensors. Data shown are mean ± S.D. from a representative experiment, with experiments performed three times. (D) Bar graph showing the basal BRET values of the WT, L267A, and F295A mutant PDE5 biosensors. Note the significant reduction in the basal BRET values of the L267A and F295A mutants compared to the WT GAFa domain. (E) Graphs showing a percentage decrease in BRET values of the WT, L267A, and F295A mutant PDE5 biosensors after 30 min incubation with the indicated cGMP concentrations. Note the decrease in the maximum %change in BRET as well as the rightward shift in the cGMP dose-response curves of the mutant biosensors. (F) Graph showing log(EC 50 ) values of cGMP-induced conformational change for the WT, L267A and F295A mutant GAFa domains in the full-length PDE5. Inset, values on top indicate the EC 50 of cGMP-induced conformational change for the respective GAFa domains in the full-length PDE5 biosensor. For (D), (E), and (F), data shown are mean ± S.D. from three experiments, with each experiment performed in triplicates. (G) Graph showing log(EC 50 ) (mean ± S.D.) values of cGMP-induced conformational change in the GAFa domain and full-length PDE5 proteins against ΔG (mean ± S.D.) values obtained from the MD simulation runs of GAFa domain (WT and mutants). (H) Cartoon representation of the miRFP670nano3 GAF domain (PDB: 7LSC) highlighting distant coevolving residues equivalent to PDE5 L267 and F295 (L100 and W125, respectively) and the ligand, biliverdin (BV). (I) Western blot analysis of the cell lysates prepared from HEK293T cells transfected with either the WT or mutant miRFP670nano3 plasmid constructs probed using an anti-FLAG antibody showing the expression of biosensor constructs. (J,K) Graphs showing relative fluorescence (J) and bioluminescence (K) of the WT, L100A and W125A mutant miRFP670nano3. Data shown are mean ± S.D. obtained from five independent experiments. p -values shown in panel (J) were obtained from Student’s t-test (unpaired, equal variance). For all tests, a p -value of <0.05 was considered statistically significant.

Article Snippet: Blots were probed using an anti-GFP antibody for GAFa and full-length PDE5 sensors (mouse IgG2a κ mAb, Santa Cruz Biotechnology, USA – SC-9996; 1:500 dilution), an anti-FLAG tag antibody for miRFP670nano3 constructs (mouse IgG2a mAB, Sino Biological, China – 109143-MM13; 1:10000 dilution), or an anti-α-tubulin antibody for α-tubulin (mouse IgG2b mAB, Proteintech, USA – 66031-1; 1:200000).

Techniques: Western Blot, Transfection, Mutagenesis, Plasmid Preparation, Construct, Expressing, Incubation, Fluorescence