Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: A, VH, VK and VL gene usage of antibodies from plasmablasts and memory B cells (MBCs). Up to the top four genes in each chart are shown with different colors (genes that were tied for 4 th and lower are not highlighted). B , Tukey’s plots showing heavy and light chain gene mutations of antibodies from plasmablasts and MBCs. Percent mutations were compared with the Mann-Whitney U-test. The middle line shows the median, and the box extends from the 1 st to 3 rd quartile. C , Top 21 neutralizing antibodies (IC50 <1 μg/mL) by antigen specificity (left), and neutralization curves of selected antibodies (right). D , SARS-CoV-2 neutralization potency versus heavy chain mutation levels of antibody panel. The 5 most potent antibodies are shown as solid circles. E , Neutralization potency of antibodies by cell type. Top values indicate percentages of non-neutralizing antibodies. Horizontal bars indicate mean values; Mann-Whitney U-test (non-neutralizing antibodies excluded from calculation). The 5 most potent antibodies are shown in color. F , SARS-CoV-2 neutralization curves of benchmark antibodies from different groups , , . G, Neutralization IC50 values of antibodies in our panel and benchmark antibodies in three different neutralization assays. Authentic SARS-CoV-2 FRNA values are from a single experiment done in quadruplicate, authentic SARS-CoV-2 (Scripps) values are an average of two experiments done in duplicate, pseudovirus (MLV) values are an average of 3 experiments in duplicate. The colors indicate the different sources of the antibodies: red, this study; blue, ref. 21; yellow, ref. 11; green, ref. 18.

Article Snippet: For screening of binding to different coronavirus antigens, streptavidin beads were coated as described above with the following biotinylated antigens: 10 µg/mL MERS spike (Sino Biological, 40069-V08B, NL63 spike (Sino Biological, 40604-V08B), 229E spike (Sino Biological, 40605- V08B), HKU1 spike (Sino Biological, 40606-V08B), OC43 spike (Sino Biological, 40607-V08B), SARS-CoV-2 NTD, SARS-CoV-1 spike, SARS-CoV-1 RBD (see above for details of production) or 10 µg/mL CD4 as a control.

Techniques: MANN-WHITNEY, Neutralization, Mutagenesis

Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: A , Isoaffinity plot of antibodies targeting SARS-CoV-2 RBD (representative of n = 2 experiments). The affinity (KD) values on the top and right of the plot refer to the dotted lines crossing the plot, e.g. any antibody falling on the 10 pM dotted line has a KD value of 10 pM. B , Neutralization potency versus affinity of anti-RBD antibodies. C , Isoaffinity plot of antibodies targeting SARS-CoV-2 NTD (representative of n = 2 experiments). The affinity (KD) values on the top and right of the plot refer to the dotted lines crossing the plot, e.g. any antibody falling on the 10 pM dotted line has a KD value of 10 pM. D , Neutralization potency versus affinity of anti-NTD antibodies. E , Epitope binning of anti-RBD antibodies (representative of n = 2 experiments). ACE2 was only used as an analyte (competitor) and not as a ligand, while all other antibodies were tested as both ligands and analytes. Solid lines indicate two-way competition while dotted lines indicate one-way competition. The number and percentage of neutralizing antibodies (IC50 < 10 µg/mL) in each bin are shown. F , Epitope bins represented by C135 (yellow bin), S309 (purple bin), ACE2 (red bin), CR3022 (cyan bin), as well as the NTD-specific antibody 4-8 (orange) modeled onto a SARS-CoV- 2 spike protein (white cartoon). Antibody 4-8 was not binned successfully in our experiments but binds to a similar region to 2-17 and 5-24 (ref. 5), which were binned. The epitope sites are color-coded the same as in and . N-glycans at the N343 glycan site are represented by sticks. G , Epitope binning of anti-NTD antibodies (representative of n = 2 experiments). All antibodies were tested as both ligands and analytes. Solid lines indicate two-way competition while dotted lines indicate one- way competition. The number and percentage of neutralizing antibodies (IC50 < 10 µg/mL) in each bin are shown. H , Binding of mAb panel to spike protein containing mutations from B.1.1.7 and B.1.351 variants (n = 1 experiment). The numbers show the percentages of mAb binding to mutants relative to D614G (which was normalized to 100). I , Neutralization potency of CV503, CV664 and CV993 against B.1.1.7 and B.1.351 variants relative to wild-type (pseudotyped) SARS-CoV-2 (n = 1 experiment). Ratios are shown in brackets, and numbers smaller than 1 indicate an increase in potency while numbers larger than 1 indicate a decrease in potency relative to wild-type.

Article Snippet: For screening of binding to different coronavirus antigens, streptavidin beads were coated as described above with the following biotinylated antigens: 10 µg/mL MERS spike (Sino Biological, 40069-V08B, NL63 spike (Sino Biological, 40604-V08B), 229E spike (Sino Biological, 40605- V08B), HKU1 spike (Sino Biological, 40606-V08B), OC43 spike (Sino Biological, 40607-V08B), SARS-CoV-2 NTD, SARS-CoV-1 spike, SARS-CoV-1 RBD (see above for details of production) or 10 µg/mL CD4 as a control.

Techniques: Neutralization, Binding Assay

Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: A , CV503 binds to the ridge region of SARS-CoV-2 RBD. The heavy and light chains of CV503 are shown in orange and yellow, respectively. SARS-CoV-2 RBD is in white, where its ridge region (residues 471–491) is shown in blue. B, The ACE2/RBD complex structure (PDB ID: 6M0J) is superimposed on the CV503/RBD complex. The heavy chain of CV503 (orange) would clash with ACE2 (green) if bound to RBD simultaneously (indicated by red circle). C-D , Epitope of CV503. Epitope residues contacting the heavy chain are in dark blue and light chain are in light blue, while residues contacting both heavy and light chains are in ocean blue. In C , CDR loops that are directly involved in RBD-binding are labeled. In D , epitope residues are labeled. Epitope residues that are also involved in ACE2 binding are labeled in red. E, ACE2-binding site on the RBD are in light pink. ACE2 is represented as semi- transparent cartoon in pale green. Epitope residues and ACE2-interacting residues are defined as those with a buried surface area (BSA) > 0 Å 2 . F , F486 at the ridge region of SARS-CoV-2 RBD (blue) is clamped in a hydrophobic pocket formed by the heavy (orange) and light chains (yellow) of CV503.

Article Snippet: For screening of binding to different coronavirus antigens, streptavidin beads were coated as described above with the following biotinylated antigens: 10 µg/mL MERS spike (Sino Biological, 40069-V08B, NL63 spike (Sino Biological, 40604-V08B), 229E spike (Sino Biological, 40605- V08B), HKU1 spike (Sino Biological, 40606-V08B), OC43 spike (Sino Biological, 40607-V08B), SARS-CoV-2 NTD, SARS-CoV-1 spike, SARS-CoV-1 RBD (see above for details of production) or 10 µg/mL CD4 as a control.

Techniques: Binding Assay, Labeling

Journal: bioRxiv

Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

doi: 10.1101/2021.04.01.437942

Figure Lengend Snippet: A , Scheme of DVD-Ig TM . In our bispecific antibody naming system, the first name refers to the antibody used to make the outer binding site and the second refers to the antibody at the inner binding site. GS or EL refers to the type of linker connecting the two antigen-binding sites. See Materials and Methods for details. B , Binding of individual and bispecific antibodies to various domains from SARS-CoV-1 and SARS-CoV-2 (representative of n = 2 experiments). Area under the curve (AUC) values are shown after subtraction with the negative control antigen. C , Neutralization potency of bispecific antibodies with SARS-CoV-2 authentic and pseudotyped virus (MLV). Values are averaged from two experiments done in duplicate. D , Neutralization curves of CV1206_521_GS with SARS-CoV-2 authentic and pseudotyped virus. Curves are from a representative experiment, IC50 values for authentic virus are the average from two experiments and those for the pseudovirus are from an average of two (bispecific) or three (regular antibody) experiments. E, Neutralization potency of CV1206_521_GS versus a cocktail of CV1206 and CV521, with concentrations shown in the molar scale to enable a fair comparison. For the antibody combination, the values on the x-axis refers to the concentration of each antibody in the cocktail, e.g. 10 nM refers to 10 nM of CV1206 + 10 nM of CV521. F , 3D reconstruction of CV1206_521_GS from nsEM images. Two “one RBD up” models (PDB 6VYB) in green are docked into the reconstruction. Similarly, multiple mock scFv’s in orange and purple were docked to approximate the DVD-Ig molecule. O, outer binding site; I, inner binding site. G , Binding of bispecific antibody panel to spike protein containing mutations from B.1.1.7 and B.1.351 variants (n = 1 experiment). The numbers show the percentages of mAb binding to mutants relative to D614G (which was normalized to 100). H , Neutralization potency of bispecific antibodies against D614G, B.1.1.7 and B.1.351 variants relative to wild-type (pseudotyped) SARS-CoV-2 (n = 1 experiment). Ratios are shown in brackets: numbers smaller than 1 indicate an increase in potency while numbers larger than 1 indicate a decrease in potency relative to wild-type. ND, not determined. I, Potency of CV503_664_EL versus individual component mAbs against wild-type and B.1.351 SARS-CoV-2 pseudotyped virus (lentivirus).

Article Snippet: For screening of binding to different coronavirus antigens, streptavidin beads were coated as described above with the following biotinylated antigens: 10 µg/mL MERS spike (Sino Biological, 40069-V08B, NL63 spike (Sino Biological, 40604-V08B), 229E spike (Sino Biological, 40605- V08B), HKU1 spike (Sino Biological, 40606-V08B), OC43 spike (Sino Biological, 40607-V08B), SARS-CoV-2 NTD, SARS-CoV-1 spike, SARS-CoV-1 RBD (see above for details of production) or 10 µg/mL CD4 as a control.

Techniques: Binding Assay, Negative Control, Neutralization, Concentration Assay

Journal: eLife

Article Title: Comprehensive analysis of nasal IgA antibodies induced by intranasal administration of the SARS-CoV-2 spike protein

doi: 10.7554/eLife.88387

Figure Lengend Snippet: ( A ) Graphs of the competitive enzyme-linked immunosorbent assay (ELISA) results showing the binding of biotinylated angiotensin-converting enzyme-2 (ACE2) to the immobilized Wuhan, Delta, or Omicron receptor binding domain (RBD) in the presence of antibodies. The results are expressed as the mean ± SD of three technical replicates. The figure reports values from a single experiment. The IC 50 values of the indicated antibodies that inhibit the RBD-ACE2 interaction are shown in the diagrams. ( B ) Comparison of neutralization activity between M-IgA and secretory IgA (S-IgA) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudotyped viruses. Neutralization curves of the indicated antibody against pseudotyped viruses bearing spike proteins of Wuhan, Delta, or Omicron are shown. Pseudotyped viruses preincubated with antibodies at the indicated concentrations were used to infect VeroE6 cells, and luciferase activities in cell lysates were measured at 20 hr post-transduction to calculate infection (%) relative to nonantibody-treated controls. The results are expressed as the mean ± SD of three technical replicates. The NT 50 values of the indicated antibodies are shown in the diagrams. Antibodies that did not reach >70% inhibition at the highest concentration tested were listed as data not determined (ND). ( C ) Comparison of neutralization potential between M-IgA and S-IgA against authentic SARS-CoV-2 BA.1. The neutralizing potential of the antibody was determined using a reverse transcription polymerase chain reaction (RT-PCR)-based SARS-CoV-2 neutralization assay. VeroE6 cells preincubated with authentic SARS-CoV-2 BA.1 virus were incubated with the indicated antibodies at various concentrations. The virus in the cell culture medium was measured at 48 hr post-transduction to calculate infection (%) relative to non-antibody-treated controls. The results are expressed as the mean ± SD of three technical replicates. The NT 50 values of the indicated antibodies are shown in the diagrams. Antibodies that did not reach >50% inhibition at the highest concentration tested are listed as ND. **p<0.01.

Article Snippet: Recombinant protein , Biotinylated SARS-CoV-2 S1 protein NTD, His, Avitag , Acro Biosystems , S1D-C52E2 , Wuhan.

Techniques: Competitive ELISA, Enzyme-linked Immunosorbent Assay, Binding Assay, Comparison, Neutralization, Activity Assay, Luciferase, Transduction, Infection, Inhibition, Concentration Assay, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Virus, Incubation, Cell Culture

Journal: Molecular Medicine Reports

Article Title: Curcumin exerts therapeutic effects on colorectal cancer by inducing pyroptosis through caspase-1 activation

doi: 10.3892/mmr.2025.13692

Figure Lengend Snippet: Cur induces pyroptosis through caspase-1 signal activation. (A and B) Cur significantly increased protein expression levels of caspase-1 and pyroptosis-related factors, while VX-765 exhibited the opposite effect. (C-F) Representative images and histogram of immunofluorescence staining of caspase-1 and GSDMD among groups. Scale bar, 40 µm. All the data are presented from three independent experiments. *P<0.05 vs. control; # P<0.05, ## P<0.01 vs. VX-765. ■ P<0.05 vs. curcumin. c-, cleaved; Cur, curcumin; GSDMD, gasdermin D; NLRP3, nucleotide-binding oligomerization domain-like receptor protein 3.

Article Snippet: Western blot analysis employed specific antibodies targeting caspase-1/3 (both precursor and cleaved forms), IL-1β, IL-18, GSDMD and nucleotide-binding oligomerization domain (NOD)-like receptor protein (NLRP)3 inflammasome components (cat. nos. ab138483, ab184787, ab2302, ab283818, ab207323, ab209845 and ab263899; Abcam; cat. no. HY- P80622 ; MedChemExpress).

Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Control, Binding Assay

Journal: PLoS Pathogens

Article Title: The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription

doi: 10.1371/journal.ppat.1008843

Figure Lengend Snippet: Full-length WT or 3L_A mutants, which mutates the conserved RLLLG motif, of Strep-tagged ORF24 (A) or homologs from MHV68 (mu24) (B), EBV (BcRF1) (C), and HCMV (UL87) (D) were transiently transfected into HEK293T cells then co-affinity purified (AP) with StrepTactinXT beads followed by western blotting. (*) indicates the presence of a non-specific band seen while using the anti-Strep antibody.

Article Snippet: To generate the plasmid for MBP-ORF24-NTD WT (Addgene #138465) and 3L_A (Addgene #138466) expression, ORF24-NTD was PCR amplified from pcDNA4/TO-ORF24-3xFLAG WT (Addgene #138423) or 3L_A (Addgene #138425) [ ] with primers to introduce SacI and BamHI sites and cloned into pMAL-c2X using T4 DNA ligase. pMAL-c2X encodes an N-terminal MBP tag and the plasmids were cloned to express ORF24-NTD with a noncleavable MBP tag.

Techniques: Transfection, Affinity Purification, Western Blot

Journal: PLoS Pathogens

Article Title: The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription

doi: 10.1371/journal.ppat.1008843

Figure Lengend Snippet: (A) Schematic of constructs used to identify a minimal N-terminal domain of ORF24 showing the predicted boundaries for the N-terminal domain, the TBP-like domain, and the C-terminal domain, including residues known to be required for Pol II binding (amino acids 73–75) and interaction with ORF34 (amino acid 328). (B) HEK293T cells were transiently transfected with full-length or truncated FLAG-tagged ORF24 and co-immunoprecipitated (IP) with anti-FLAG beads followed by western blotting with the indicated antibodies to detect ORF24 and Pol II. (*) indicates the presence of a non-specific band seen while using the anti-Strep antibody.

Article Snippet: To generate the plasmid for MBP-ORF24-NTD WT (Addgene #138465) and 3L_A (Addgene #138466) expression, ORF24-NTD was PCR amplified from pcDNA4/TO-ORF24-3xFLAG WT (Addgene #138423) or 3L_A (Addgene #138425) [ ] with primers to introduce SacI and BamHI sites and cloned into pMAL-c2X using T4 DNA ligase. pMAL-c2X encodes an N-terminal MBP tag and the plasmids were cloned to express ORF24-NTD with a noncleavable MBP tag.

Techniques: Construct, Binding Assay, Transfection, Immunoprecipitation, Western Blot

Journal: PLoS Pathogens

Article Title: The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription

doi: 10.1371/journal.ppat.1008843

Figure Lengend Snippet: (A-D) Full-length or truncated Strep-tagged constructs of ORF24. (A) or homologs from MHV68 (mu24; B), EBV (BcRF1; C), and HCMV (UL87; D) were transiently transfected into HEK293T cells then co-affinity purified (AP) with StrepTactinXT beads followed by western blotting. (*) indicates the presence of a non-specific band seen while using the anti-Strep antibody.

Article Snippet: To generate the plasmid for MBP-ORF24-NTD WT (Addgene #138465) and 3L_A (Addgene #138466) expression, ORF24-NTD was PCR amplified from pcDNA4/TO-ORF24-3xFLAG WT (Addgene #138423) or 3L_A (Addgene #138425) [ ] with primers to introduce SacI and BamHI sites and cloned into pMAL-c2X using T4 DNA ligase. pMAL-c2X encodes an N-terminal MBP tag and the plasmids were cloned to express ORF24-NTD with a noncleavable MBP tag.

Techniques: Construct, Transfection, Affinity Purification, Western Blot

Journal: PLoS Pathogens

Article Title: The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription

doi: 10.1371/journal.ppat.1008843

Figure Lengend Snippet: (A) Colloidal Coomassie gel demonstrating that GST and GST-ORF24-NTD can be recombinantly expressed in E. coli and purified by glutathione sepharose purification. (B) GST or GST-ORF24-NTD was incubated in HEK293T whole cell lysate, then subjected to affinity purification using glutathione magnetic beads (GSH AP) followed by western blotting. (C) Sequential reconstitution strategy for a minimal PIC containing GST-ORF24-NTD. (D) Representative reference-free two-dimensional class averages of negatively stained minimal PICs (TBP/TFIIA/TFIIB/TFIIF/Pol II/DNA) assembled in the presence of GST-ORF24-NTD. Three classes on the left show different views of the minimal PIC alone, with the class average in the upper-left annotated with the main features of a minimal PIC particle. The nine class averages on the right show diffuse density in various positions around the Pol II stalk attributed to bound GST-ORF24-NTD (green arrows). (E) Representative three-dimensional class averages of negatively stained minimal PICs assembled in the presence of GST-ORF24-NTD. Classes 1 and 2 exhibit two major areas occupied by bound GST-ORF24-NTD proximal to the Pol II stalk, while class 5 does not exhibit any such density near the Pol II stalk. Solid surfaces are colored by subunit, while a lower intensity iso-surface is shown in transparency to reveal the weaker density attributed to bound GST-ORF24-NTD (green arrows). (F) Difference mapping of the densities attributed to bound GST-ORF24-NTD. Shown on the left are two-dimensional projections of class 1 (top) and 2 (bottom) from (E), and on the right are the difference maps, called “Class 1 Δ” and “Class 2 Δ”, calculated by subtracting Class 5 from each of the respective classes. (G) Three-dimensional difference maps corresponding to the extra density attributed to bound GST-ORF24-NTD, mapped onto the structure of the minimal PIC (PDB 5IYA). (H) Zoomed in view of (G) with the structure of Schizosaccharomyces pombe Rpb1 (PDB 3H0G) superposed onto the human structure to show the location of the beginning portion of the Rpb1 CTD within the Pol II stalk. Note that only the very N-terminal portion of the Rpb1 CTD is visible in this structure, with >450 amino acids following this sequence in the CTD of human Rpb1.

Article Snippet: To generate the plasmid for MBP-ORF24-NTD WT (Addgene #138465) and 3L_A (Addgene #138466) expression, ORF24-NTD was PCR amplified from pcDNA4/TO-ORF24-3xFLAG WT (Addgene #138423) or 3L_A (Addgene #138425) [ ] with primers to introduce SacI and BamHI sites and cloned into pMAL-c2X using T4 DNA ligase. pMAL-c2X encodes an N-terminal MBP tag and the plasmids were cloned to express ORF24-NTD with a noncleavable MBP tag.

Techniques: Purification, Incubation, Affinity Purification, Magnetic Beads, Western Blot, Staining, Sequencing

Journal: PLoS Pathogens

Article Title: The gammaherpesviral TATA-box-binding protein directly interacts with the CTD of host RNA Pol II to direct late gene transcription

doi: 10.1371/journal.ppat.1008843

Figure Lengend Snippet: (A) Recombinantly purified GST-CTD repeats or the GST-CTD linker were incubated with either purified MBP-ORF24-NTD or MBP-ORF24-NTD-3L_A protein, then subjected to a glutathione pulldown (GSH AP). Samples were resolved by SDS-PAGE and analyzed by western blot. (B) Recombinantly purified GST-CTD repeats or GST-Rpb4/His-Rpb7 heterodimer were incubated with recombinantly purified Strep-tagged ORF24-NTD, then subjected to a StrepTactinXT pulldown (AP). Samples were resolved by SDS-PAGE and analyzed by western blot. (C) Recombinantly purified full-length GST-CTD repeats (52 repeats) shorter GST-CTD constructs (10, 4, or 2 repeats), or GST itself were incubated with recombinantly purified Strep-tagged ORF24-NTD, then subjected to a StrepTactinXT pulldown (AP). Samples were resolved by SDS-PAGE and stained with colloidal Coomassie.

Article Snippet: To generate the plasmid for MBP-ORF24-NTD WT (Addgene #138465) and 3L_A (Addgene #138466) expression, ORF24-NTD was PCR amplified from pcDNA4/TO-ORF24-3xFLAG WT (Addgene #138423) or 3L_A (Addgene #138425) [ ] with primers to introduce SacI and BamHI sites and cloned into pMAL-c2X using T4 DNA ligase. pMAL-c2X encodes an N-terminal MBP tag and the plasmids were cloned to express ORF24-NTD with a noncleavable MBP tag.

Techniques: Purification, Incubation, SDS Page, Western Blot, Construct, Staining

Journal: Scientific Reports

Article Title: Anti-nucleocapsid antibodies enhance the production of IL-6 induced by SARS-CoV-2 N protein

doi: 10.1038/s41598-022-12252-y

Figure Lengend Snippet: SARS-CoV-2 N protein, but not S protein, induced IL-6 production. The levels of IL-6 were measured by ELISA 2 days after treatment ( A , C , and F ). ( A ) K-ML2 cells were stimulated with the lysate of 293 T cells transfected with plasmids encoding each of the SARS-CoV-2 proteins. ( B ) The expression of each viral protein confirmed by western blot using the anti-Strep tag. The images from three membranes were combined. The original images with different exposure time are shown in Fig. . ( C ) K-ML2 cells were stimulated with serially diluted S or N protein produced using a baculovirus expression system. The mean and standard deviation of triplicate samples are shown. ( D ) A schematic diagram of full-length and truncated N proteins. ( E ) Anti-NTD (N2) antibody and anti-CTD (C2) antibody were used to visualize the full-length and truncated N proteins by western blot. The images from two membranes transferred from a single gel were combined. A blue vertical dividing line of two membranes is shown. The original images with different exposure time are shown in Fig. . ( F ) K-ML2 cells were stimulated with the cell lysates of 293 T cells expressing the full-length or truncated N proteins. Data are expressed as the mean and standard deviation of triplicate samples. The representative results of three independent experiments are shown.

Article Snippet: Ninety-six-well flat-bottom microplates were coated with 100 ng/well of N protein (40588-V08B, Sino Biological), NTD (40588-V07E10, Sino Biological), or CTD (40588-V07E5, Sino Biological) in 50 μL of carbonate-bicarbonate buffer (C-3041, Sigma, St. Louis, MO), and incubated at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Western Blot, Strep-tag, Produced, Standard Deviation

Journal: Scientific Reports

Article Title: Anti-nucleocapsid antibodies enhance the production of IL-6 induced by SARS-CoV-2 N protein

doi: 10.1038/s41598-022-12252-y

Figure Lengend Snippet: Anti-N antibodies and COVID-19 patient sera enhanced the IL-6 production induced by N protein. Levels of IL-6 were measured by ELISA 2 days after the addition of N protein, except for in panel C. ( A ) K-ML2 cells were stimulated with 156 ng/mL of N protein with escalating amounts of anti-S (blue) or anti-N (red) antibodies. ( B ) Anti-N antibody, S2, was digested by pepsin, and purified F(ab’)2 (red) or IgG (blue) was added to induce IL-6 production. Data are expressed as the mean and standard deviation of triplicate samples. ( C ) The levels of mouse mAbs bound to the full-length N protein (blue), NTD (Thr49-Gly175 fragment: red) or CTD (Lys248-Pro365 fragment: gray) were measured by an in-house ELISA. Anti-S mAb, clone 29-C7, was used as a negative control. ( D ) K-ML2 cells were stimulated with 3 μL of the cell lysate of N protein-expressing 293 T cells in the presence of 1 μg/mL of mAbs. Data are expressed as the mean and standard deviation of triplicate samples. The representative results of three independent experiments are shown. ( E ) K-ML2 cells were stimulated with 156 ng/mL of N protein with escalating amounts of serum from two patients, Patient 313 (blue) and Patient 315 (red). “0%” indicates the baseline production of IL-6 induced by N protein without patients’ serum. “10% noN” indicates the level of non-specific IL-6 induction from the addition of 10% patient serum from patient No. 313 without N protein. ( F ) The levels of IL-6 in the culture supernatants of K-ML2 cells cultured in the presence of 1% patient serum. Specimens were divided into four groups according to the disease severity at the time of blood collection. The criteria for COVID-19 severity in Japan were described in the Methods section. In brief, the criteria for mild, moderate I, moderate II, and severe are almost the same as those of mild, moderate, severe, and critical in the US NIH guideline. The center lines in the boxes and the boxes indicate the median and 25/75 percentiles, respectively. “*” denotes a statistically significant difference among the four groups ( p < 0.0001) by the Kruskal–Wallis test. “**” and “***” denote a statistically significant difference between the moderate I and severe disease groups ( p < 0.0001) and between the mild and severe disease groups ( p < 0.0001), respectively, by the Mann–Whitney U test.

Article Snippet: Ninety-six-well flat-bottom microplates were coated with 100 ng/well of N protein (40588-V08B, Sino Biological), NTD (40588-V07E10, Sino Biological), or CTD (40588-V07E5, Sino Biological) in 50 μL of carbonate-bicarbonate buffer (C-3041, Sigma, St. Louis, MO), and incubated at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Standard Deviation, Negative Control, Expressing, Cell Culture, MANN-WHITNEY

Journal: Signal Transduction and Targeted Therapy

Article Title: SARS-CoV-2 envelope protein impairs airway epithelial barrier function and exacerbates airway inflammation via increased intracellular Cl − concentration

doi: 10.1038/s41392-024-01753-z

Figure Lengend Snippet: SARS-CoV-2 envelope (E) protein disrupted barrier function and altered the expression of tight junction proteins in airway epithelial cells. a , b 16HBE14o- monolayers were stimulated with E protein (50 μg/ml) apically for the indicated time points. a Transepithelial resistance (TER) values were measured and shown relative to the TER before E protein stimulation ( n = 5). b Fluorescence intensity of fluorescein isothiocyanate-dextran (4 kDa, FD4) flux across cell monolayers were monitored ( n = 3). c Diagram of Pseudomonas aeruginosa strain PAO1 penetration through airway epithelial cell monolayer. Airway epithelial cells were seeded into the upper chamber of plate inserts with a 3 μm pore size membrane, which allowed the passage of PAO1. Cell monolayer with tight junctions (TJs) were infected apically with PAO1 and the penetrated bacteria from the basolateral medium in different groups were enumerated. The figure was created using Adobe Illustrator. d The colony-forming units (CFU) of penetrated PAO1 obtained from the plate counting technique. 16HBE14o- monolayers were infected with PAO1 at a multiplicity of infection (MOI) of 20 for 2 h, with or without E protein (50 μg/ml) stimulation ( n = 3). e ICR mice were injected with FD4 after the stimulation of E protein (300 μg/ml) or saline for 6 h. The fluorescence ratio of BALF and serum was calculated ( n = 6). f Heatmap of tight junction-related genes with significant changes in BEAS-2B cells stimulated with E protein (50 μg/ml) for 12 h. g Quantitative real-time PCR analyses showing the expression of ZO-1, occludin and claudin-4 in BEAS-2B cells stimulated with E protein (50 μg/ml) for the indicated time points ( n = 3). Data are shown as means ± S.D. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the control group. h Representative immunoblots showing the expression level of ZO-1, occludin and claudin-4 in BEAS-2B cells after stimulation with E protein (50 μg/ml) for the indicated time points. GAPDH served as a loading control. i Immunofluorescence images showing the expression of claudin-4 in BEAS-2B cells, in the absence or presence of E protein (50 μg/ml) stimulation. Scale bar = 20 μm. j Immunofluorescence staining of lung sections showing the expression of claudin-4 in Ad5-hACE2 transgenic mice with or without SARS-CoV-2 infection (1 × 10 5 PFU). Scale bar = 50 μm. k After incubation with E protein (50 μg/ml), the co-immunoprecipitates and the total lysates (Input) were analyzed by immunoblotting with the indicated antibodies

Article Snippet: The antibodies against SARS-CoV-2 Spike S1 (1:1000, #40591-R235) and Spike S2 (1:1000, #40590-T62) were from Sino Biological (China).

Techniques: Expressing, Fluorescence, Pore Size, Membrane, Infection, Bacteria, Injection, Saline, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Transgenic Assay, Incubation

Journal: Signal Transduction and Targeted Therapy

Article Title: SARS-CoV-2 envelope protein impairs airway epithelial barrier function and exacerbates airway inflammation via increased intracellular Cl − concentration

doi: 10.1038/s41392-024-01753-z

Figure Lengend Snippet: SARS-CoV-2 envelope (E) protein induced robust inflammation in airway epithelial cells. a Quantitative real-time PCR analyses showing the expression of pro-inflammatory cytokines in BEAS-2B cells stimulated with E protein (50 μg/ml) for the indicated time ( n = 3). b A volcano plot from RNA sequencing analysis illustrating the distribution of all differentially expressed genes (DEGs) in BEAS-2B cells with and without E protein (50 μg/ml) stimulation. c Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of DEGs in BEAS-2B cells stimulated with E protein (50 μg/ml) for 12 h. The dot size represents the number of DEGs, while the dot’s color corresponds to the P value. d Quantitative real-time PCR analyses showing the expression of pro-inflammatory cytokines or chemokines in the lung tissues of mice after intratracheal instillation of different concentrations of E protein for 24 h ( n = 3–9). Data are shown as means ± S.D. ns = no significant, * P < 0.05, ** P < 0.01, *** P < 0.001 versus the control group. e Hematoxylin and eosin (HE) staining of lung samples from mice after intratracheal instillation of E protein (100 μg/ml) for 24 h. Scale bar = 50 μm

Article Snippet: The antibodies against SARS-CoV-2 Spike S1 (1:1000, #40591-R235) and Spike S2 (1:1000, #40590-T62) were from Sino Biological (China).

Techniques: Real-time Polymerase Chain Reaction, Expressing, RNA Sequencing Assay, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: SARS-CoV-2 envelope protein impairs airway epithelial barrier function and exacerbates airway inflammation via increased intracellular Cl − concentration

doi: 10.1038/s41392-024-01753-z

Figure Lengend Snippet: SARS-CoV-2 envelope (E) protein induced airway inflammation via the TLR2/4-JNK-AP-1 signaling pathway. a Representative immunoblots showing the phosphorylation level of p38, ERK and JNK in BEAS-2B cells after E protein (50 μg/ml) stimulation for 12 h. GAPDH served as a loading control. b Immunofluorescence staining of lung sections showing the phosphorylation of JNK in Ad5-hACE2 transgenic mice with or without SARS-CoV-2 infection (1 × 10 5 PFU). Scale bar = 50 μm. c Co-immunoprecipitates and the total lysates (Input) were detected by Western Blotting using anti-TLR2 and anti-TLR4 antibody. d GST pull-down assays using GST-E protein and TLR2. TLR2 binding to GST-E protein was determined by using an anti-His antibody. e GST pull-down assays using GST-E protein and TLR4. TLR4 binding to GST-E protein was determined by using an anti-His antibody. f Representative immunoblots showing the effect of C29 (50 μM), an inhibitor of TLR2, on the phosphorylation level of JNK in BEAS-2B cells after stimulation with E protein (50 μg/ml) for 12 h. GAPDH served as a loading control. g Representative immunoblots showing the effect of Resatorvid (5 μM), an inhibitor of TLR4, on the phosphorylation level of JNK in BEAS-2B cells after stimulation with E protein (50 μg/ml) for 12 h. GAPDH served as a loading control. h Representative immunoblots showing the phosphorylation level of c-Jun in BEAS-2B cells after E protein (50 μg/ml) stimulation for 12 h. GAPDH served as a loading control. i Representative immunoblots showing the effect of SP600125 (10 μM), the selective inhibitor of JNK, on the phosphorylation level of JNK in BEAS-2B cells after stimulation with E protein (50 μg/ml) for 12 h. GAPDH served as a loading control. j Heatmap showing significant changes in mRNA expression of inflammation-related genes in BEAS-2B cells stimulated with E protein (50 μg/ml) with or without SP600125 (10 μM) for 12 h ( n = 3/group). k Quantitative real-time PCR analyses showing the effect of C29 (50 μM), SP600125 (10 μM) and the selective AP-1 inhibitor T-5224 (10 μM) on the expression of tumor necrosis factor-α (TNF-α) in BEAS-2B cells stimulated with E protein (50 μg/ml) for 12 h ( n = 3). l Quantitative real-time PCR analyses showing the effect of SP600125 (30 mg/kg) on the expression of TNF-α in the lung samples from mice after intratracheal instillation of E protein (100 μg/ml) for 24 h ( n = 5). Data are shown as means ± S.D. ** P < 0.01, *** P < 0.001 indicated by lines. m Hematoxylin and eosin (HE) staining of lung samples from mice after intratracheal instillation of E protein (100 μg/ml) for 24 h, with or without intraperitoneal pre-treatment of SP600125 (30 mg/kg). Scale bar = 50 μm

Article Snippet: The antibodies against SARS-CoV-2 Spike S1 (1:1000, #40591-R235) and Spike S2 (1:1000, #40590-T62) were from Sino Biological (China).

Techniques: Western Blot, Immunofluorescence, Staining, Transgenic Assay, Infection, Binding Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: Signal Transduction and Targeted Therapy

Article Title: SARS-CoV-2 envelope protein impairs airway epithelial barrier function and exacerbates airway inflammation via increased intracellular Cl − concentration

doi: 10.1038/s41392-024-01753-z

Figure Lengend Snippet: SARS-CoV-2 envelope (E) protein induced elevated intracellular Cl − concentration ([Cl − ] i ) through TLR2-JNK-AP-1 pathway in airway epithelial cells. a Representative trace showing the short-circuit current ( I SC ) response induced by apical (ap) administration of forskolin (10 μM) with (right panel) or without (left panel) E protein (50 μg/ml) stimulation for 24 h in 16HBE14o- cells. b The effect of E protein (50 μg/ml) on the I SC currents elicited by forskolin (10 μM, ap) in 16HBE14o- cells. ( n = 6–8). c [Cl − ] i was measured in BEAS-2B cells stimulated with E protein (50 μg/ml) for the indicated time points. ( n = 15–30 cells for each group). d Representative immunoblots showing the expression level of cystic fibrosis transmembrane conductance regulator (CFTR) in BEAS-2B cells after E protein (50 μg/ml) stimulation for the indicated time points. GAPDH served as a loading control. e Quantitative real-time PCR analyses showing the expression of phosphodiesterase 4D (PDE4D) in BEAS-2B cells stimulated with E protein (50 μg/ml) for the indicated time points ( n = 3). f Effect of E protein (50 μg/ml) on the intracellular level of cAMP in BEAS-2B cells ( n = 3). g Effects of the TLR2 inhibitor C29 (50 μM), the TLR4 inhibitor Resatorvid (5 μM), the JNK inhibitor SP600125 (10 μM) and the AP-1 inhibitor T-5224 (10 μM) on the expression of PDE4D in BEAS-2B cells after E protein (50 μg/ml) stimulation for 12 h ( n = 3). h [Cl − ] i was measured in BEAS-2B cells stimulated with E protein (50 μg/ml) for 12 h, with or without pre-treatment with C29 (50 μM), Resatorvid (5 μM), SP600125 (10 μM) or T-5224 (10 μM). ( n = 15–40 cells for each group). i [Cl − ] i was measured in primary cultured mouse airway epithelial cells (mPAECs) stimulated with E protein (50 μg/ml) for 24 h ( n = 18–20 cells for each group). j Quantitative real-time PCR analyses showing the expression of PDE4D in the mPAECs stimulated with E protein (100 μg/ml) for 24 h ( n = 3). Data are shown as means ± S.D. ns = no significant, * P < 0.05, ** P < 0.01, *** P < 0.001 versus the control group or indicated by lines. k Immunofluorescence images showing the expression of PDE4D in Ad5-hACE2 transgenic mice with or without SARS-CoV-2 infection (1 × 10 5 PFU). Scale bar = 50 μm

Article Snippet: The antibodies against SARS-CoV-2 Spike S1 (1:1000, #40591-R235) and Spike S2 (1:1000, #40590-T62) were from Sino Biological (China).

Techniques: Concentration Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Immunofluorescence, Transgenic Assay, Infection

Journal: Signal Transduction and Targeted Therapy

Article Title: SARS-CoV-2 envelope protein impairs airway epithelial barrier function and exacerbates airway inflammation via increased intracellular Cl − concentration

doi: 10.1038/s41392-024-01753-z

Figure Lengend Snippet: SARS-CoV-2 envelope (E) protein induced Cl − -driven airway inflammation via activating serum/glucocorticoid regulated kinase 1 (SGK1). a Representative immunoblots showing the phosphorylation level of IκB and SGK1 in BEAS-2B cells after E protein (50 μg/ml) stimulation for the indicated time points. GAPDH served as a loading control. b Representative immunoblots showing the effect of SGK1 knockout (KO) on the phosphorylation level of IκB in BEAS-2B cells after E protein (50 μg/ml) stimulation for the indicated time points. GAPDH served as a loading control. c Quantitative real-time PCR analyses showing the effect of SGK1 KO on the expression of tumor necrosis factor-α (TNF-α) in BEAS-2B cells stimulated with E protein (50 μg/ml) for the indicated time points ( n = 3). d Representative immunoblots showing the phosphorylation level of SGK1 in lung samples derived from mice after intratracheal instillation of E protein (100 μg/ml) for 24 h. GAPDH served as a loading control. e Representative immunoblots showing the effect of EMD638683 (10 mg/kg), a selective inhibitor of SGK1, on the phosphorylation level of IκB in lung samples derived from mice after intratracheal instillation of E protein (100 μg/ml) for 24 h. GAPDH served as a loading control. f Quantitative real-time PCR analyses showing the effect of EMD638683 (10 mg/kg) on the expression of TNF-α in the lung samples from mice after intratracheal instillation of E protein (100 μg/ml) for 24 h ( n = 3–8). Data are shown as means ± S.D. * P < 0.05, *** P < 0.001 versus the control group or indicated by lines. g Hematoxylin and eosin (HE) staining of lung samples from mice after intratracheal instillation of E protein (100 μg/ml) for 24 h, with or without intraperitoneal pre-treatment of EMD638683 (10 mg/kg). Scale bar = 50 μm

Article Snippet: The antibodies against SARS-CoV-2 Spike S1 (1:1000, #40591-R235) and Spike S2 (1:1000, #40590-T62) were from Sino Biological (China).

Techniques: Western Blot, Knock-Out, Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: SARS-CoV-2 envelope protein impairs airway epithelial barrier function and exacerbates airway inflammation via increased intracellular Cl − concentration

doi: 10.1038/s41392-024-01753-z

Figure Lengend Snippet: Phosphodiesterase 4 (PDE4) inhibitor protected against SARS-CoV-2 envelope (E) protein-induced airway inflammation through suppressing intracellular Cl − accumulation. a Intracellular Cl − concentration ([Cl − ] i ) was measured in BEAS-2B cells stimulated with E protein (50 μg/ml) for the indicated time points, with or without pre-treatment of rolipram (20 μM), a selective PDE4 inhibitor ( n = 21–30 cells for each group). b Representative immunoblots showing the effect of rolipram (20 μM) on the phosphorylation level of IκB in BEAS-2B cells after E protein (50 μg/ml) stimulation for the indicated time points. GAPDH served as a loading control. c Quantitative real-time PCR analyses showing the effect of rolipram (20 μM) on the expression of tumor necrosis factor-α (TNF-α) in BEAS-2B cells stimulated with E protein (50 μg/ml) for the indicated time points ( n = 3). d [Cl − ] i was measured in the primary cultured mouse airway epithelial cells (mPAECs) stimulated with E protein (50 μg/ml) for 24 h, with or without pre-treatment of rolipram (20 μM) ( n = 19–20 cells for each group). e Representative immunoblots showing the effect of rolipram (10 mg/kg) on the phosphorylation level of IκB in lung samples from mice after intratracheal instillation of E protein (100 μg/ml) for 24 h. GAPDH served as a loading control. f Quantitative real-time PCR analyses showing the effect of rolipram (10 mg/kg) on the expression of TNF-α in the lung samples derived from mice after intratracheal instillation of E protein (100 μg/ml) for 24 h ( n = 5–6). Data are shown as means ± S.D. ** P < 0.01, *** P < 0.001 indicated by lines. g Hematoxylin and eosin (HE) staining of the lung samples from mice after intratracheal instillation of E protein (100 μg/ml) for 24 h, with or without intraperitoneal pre-treatment of rolipram (10 mg/kg). Scale bar = 50 μm

Article Snippet: The antibodies against SARS-CoV-2 Spike S1 (1:1000, #40591-R235) and Spike S2 (1:1000, #40590-T62) were from Sino Biological (China).

Techniques: Concentration Assay, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Derivative Assay, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: SARS-CoV-2 envelope protein impairs airway epithelial barrier function and exacerbates airway inflammation via increased intracellular Cl − concentration

doi: 10.1038/s41392-024-01753-z

Figure Lengend Snippet: SARS-CoV-2 envelope (E) protein down-regulated tight junction proteins and triggered inflammation and intracellular Cl − accumulation in human primary cultured airway epithelial cells (hPAECs). a Representative immunoblots showing the expression level of ZO-1, occludin and claudin-4 in hPAECs stimulated with E protein (50 μg/ml) for 6 h. GAPDH served as a loading control. b Immunofluorescence images showing the expression of ZO-1 in hPAECs, in the absence or presence of E protein stimulation for 6 h. c Quantitative real-time PCR analyses showing the expression of pro-inflammatory cytokines in hPAECs stimulated with E protein (50 μg/ml) for 12 h ( n = 3). d Representative immunoblots showing the expression of cystic fibrosis transmembrane conductance regulator (CFTR) and the phosphorylation level of JNK, serum/glucocorticoid regulated kinase 1 (SGK1) and IκB in hPAECs after stimulation with the E protein (50 μg/ml) for 12 h. GAPDH served as a loading control. e Immunofluorescence images showing the phosphorylation of JNK in hPAECs, in the absence or presence of E protein (50 μg/ml) stimulation. f Immunofluorescence images showing the phosphorylation of SGK1 in hPAECs, in the absence or presence of E protein (50 μg/ml) stimulation. g Quantitative real-time PCR analyses showing the expression of phosphodiesterase 4 (PDE4) in hPAECs stimulated with E protein (50 μg/ml) for the indicated different time points ( n = 3). h Intracellular Cl − concentration ([Cl − ] i ) was measured in hPAECs stimulated with E protein (50 μg/ml) for 12 h ( n = 25 cells for each group). Data are shown as means ± S.D. ** P < 0.01, *** P < 0.001 versus the control group

Article Snippet: The antibodies against SARS-CoV-2 Spike S1 (1:1000, #40591-R235) and Spike S2 (1:1000, #40590-T62) were from Sino Biological (China).

Techniques: Cell Culture, Western Blot, Expressing, Immunofluorescence, Real-time Polymerase Chain Reaction, Concentration Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: SARS-CoV-2 envelope protein impairs airway epithelial barrier function and exacerbates airway inflammation via increased intracellular Cl − concentration

doi: 10.1038/s41392-024-01753-z

Figure Lengend Snippet: Schematic diagram showing the pathophysiological role of SARS-CoV-2 envelope (E) protein in airway epithelial cells. SARS-CoV-2 E protein down-regulated tight junctional proteins, impairing airway epithelial integrity. E protein also activated TLR2/4-JNK-AP-1 signaling, leading to increased PDE4 expression and reduced cAMP levels. This disruption of cAMP signaling impaired CFTR-mediated Cl − transport, resulting in elevated [Cl − ] i and activation of SGK1, which contributes to the ongoing inflammation in the airway epithelium

Article Snippet: The antibodies against SARS-CoV-2 Spike S1 (1:1000, #40591-R235) and Spike S2 (1:1000, #40590-T62) were from Sino Biological (China).

Techniques: Expressing, Disruption, Activation Assay