Journal: Cell Reports Medicine

Article Title: Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

doi: 10.1016/j.xcrm.2025.102035

Figure Lengend Snippet:

Article Snippet: For SARS-CoV-2 spike binding ELISAs in B–7E, assays were performed as above, except for coating with the following proteins where indicated: SARS-CoV-2 wild type spike RBD (Cat# 40592-V08H, Sino Biologicals), SARS-CoV-2 D614G spike (Cat# 40589-V08H8, Sino Biologicals), SARS-CoV-2 B.1.617.2 spike (Cat# 40589-V08H10, Sino Biologicals), SARS-CoV-2 B.1.1.529 spike (Cat# 40589-V08H26), or SARS-CoV-2 BA.2 spike (Cat# 40589-V08H28, Sino Biologicals).

Techniques: Virus, Recombinant, Lysis, Reporter Gene Assay, Cell Stimulation, Electron Microscopy, Luminex, Enzyme-linked Immunospot, Luciferase, Plasmid Preparation, Software, Synthesized

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: A bi-allelic splicing donor site variant in LRRC23 was identified from asthenozoospermia patients. ( A ) A consanguineous pedigree with two infertile males (IV-1 and IV-2). IV-1 was subjected for WES (arrow). Genotypes of the variant (blue) in all attended family members (III-1, III-2, IV-1, IV-2, IV-3, and IV-4) are confirmed by Sanger sequencing. +, wild-type allele. An infertile female sibling (IV-4) is marked in black circle. ( B ) Papanicolaou-stained sperm from the infertile male (IV-2). ( C ) Mapping of the LRRC23 variant. Mutation of G to A at the splicing donor site in the 5 th intron is predicted to prevent LRRC23 mRNA from splicing. ( D ) Sequencing chromatograms presenting the LRRC23 variant in the infertile male (IV-1) and his father (III-2). The variant is underlined and normal splicing donor site (GT) is boxed. ( E-F ) Minigene assay for testing altered splicing of LRRC23 by the variant. ( E ) Minigene constructs expressing LRRC23 ORF containing the 5 th intron (sashed) with wild-type (WT) or mutant (Mut, red) splicing donor site were generated. The constructs are tagged with FLAG and HA at N- and C-termini, respectively. ( F ) RT-PCR of the 293T cells transfected with the minigene constructs reveals the 5 th intron is not spliced out and retained by the variant. Intron-spanning primers, F1 and R1, are used. Three times biological replicated.

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Variant Assay, Sequencing, Staining, Mutagenesis, Mini Gene Assay, Construct, Expressing, Generated, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: Lrrc23 mutant mice mimicking human splice variant phenocopy male infertility and reduced sperm motility. ( A-B ) Immunoblotting of LRRC23 in testis ( A ) and epididymal sperm ( B ) from mutant male mice. Truncated LRRC23 (arrowheads) is detected from testis microsome fraction (filled), but not in mature sperm (empty), of heterozygous (+/Δ) and homozygous (Δ/Δ) males. Acetylated tubulin (AcTub) is a loading control. ( C ) Confocal images of immunostained LRRC23 in Lrrc23 +/Δ and Lrrc23 Δ/Δ epididymal sperm. Samples from WT were used for positive or negative control of normal or truncated LRRC23 ( A, B , and C ). ( D ) Epididymal sperm counts. n.s., not significant. ( E ) Pregnancy rate of Lrrc23 +/Δ and Lrrc23 Δ/Δ males. ( F ) Number of litters from fertile females mated with Lrrc23 +/Δ and Lrrc23 Δ/Δ males. ( G ) Swimming trajectory of Lrrc23 +/Δ and Lrrc23 Δ/Δ sperm in viscous media (0.3% methylcellulose). Swimming trajectory for 2 seconds is overlaid. ( H ) Flagellar waveforms of Lrrc23 +/Δ and Lrrc23 Δ/Δ sperm before (0 minute) and after (90 minutes) inducing capacitation. Flagellar movements for two beat cycles are overlaid and color coded in time. Circles indicate sperm counts from individual males ( D ) and pup numbers from each litter ( F ). Data represented as mean ± SEM ( D and F ). Statistical comparison was perfomed by Mann-whiteny U test (D) or Student’s t-test (F). Experiments were repeated three times with biological replications ( A, B, C, G , and H ).

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Mutagenesis, Variant Assay, Western Blot, Control, Negative Control, Comparison

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: C-terminal truncation of human LRRC23 by the splicing site mutation prevents its interaction with radial spoke (RS) head. ( A ) Sub-tomogram averaging images of RSs from Chlamydomonas reinhardtii ( left ), Trypanosoma brucei ( middle ), and mouse sperm ( right ). Original data from Electron Microscopy Data Bank was rendered. ( B ) Structure of RS in C. reinhardtii . A schematic cartoon shows the RS1 and 2. The structure of RS2 stalk is shown in inset (PDB Id: 7JRJ). ( C - D ) Purification of normal (hLRRC23 WT ) and the mutant human LRRC23 (hLRRC23 Mut ) by the splicing site mutation (c.621+1G>A) in this study. ( C ) Diagrams for the purified recombinant normal and mutant proteins tagged with tagged with GST and HA at N- and C-termini, respectively. ( D ) Purified proteins by Coomassie blue staining ( left ) and immunoblotting with a-HA ( middle ) and a-LRRC23 ( right ). Proteins matched to the predicted size were marked with asterisks. ( E ) A cartoon of the RSPH-trap approach to test LRRC23 interaction with RS proteins. Individual human RS proteins tagged with FLAG (RSPH-FLAG) are expressed in 293T cells and enriched by α-FLAG resin from cell lysates. The recombinant RSPH proteins were incubated with the purified hLRRC23 WT or hLRRC23 Mut and subjected to immunoblotting. ( F ) Interaction of hLRRC23 to a RS head component, RSPH9. The purified hLRRC23 were incubated with the RSPH-Trap (RS head, RSPH6A and RSPH9; stalk, RSPH3 and RSPH22) and subjected to immunoblotting. 5% amount of the hLRRC23s used for the trap assay were loaded as inputs. White lines in individual α-HA blot images indicate marker information (75 kDa, left ; 50 kDa, right ). Experiments were repeated four times. Purified GST was used for negative control ( SI Appendix , Fig. S4 B ). ( G ) A phylogenetic tree constructed by Maximum-likelihood analysis of the protein sequences of the C. reinhardtii RSP15 and the orthologs of LRRC23 and LRRC34. LRR37, the first LRRC23 ortholog identified in Ciona intestinalis is marked in bold. ( H ) Comparison of the reported RSP15 from C. reinhardtii and the predicted structure of LRRC23 and LRRC34 from human. Atomic structure of the C. reinhardtii RS2 containing RSP15 are represented by ribbon (RS2) and surface (RSP15) diagram ( left , PDB Id: 7JU4). Ribbon diagrams of C. reinhardtii RSP15 and AlphaFold-predicted human LRRC23 ( middle ) and LRRC34 ( right ) are shown for structural comparison. Secondary structures are color-coded. Different from C. reinhardtii RSP15 and LRRC34, LRRC23 does not display repeated α-helix (magenta) between β-sheets (gold).

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Mutagenesis, Electron Microscopy, Purification, Recombinant, Staining, Western Blot, Incubation, TRAP Assay, Marker, Negative Control, Construct, Comparison

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: Head of the third radial spoke is absent in Lrrc23 Δ/Δ sperm flagella. ( A - B ) Sub-tomogram averaging (STA) to analyze structural defects at radial spoke (RS) of WT ( A ) and Lrrc23 Δ/Δ sperm ( B ). Shown are STA images resulted from 96-nm doublet repeats from WT and Lrrc23 Δ/Δ sperm. RS2 and 3 are magnified and density to represent RS3 head and the bridge between RS2 and RS3 (red circle) is missed in Lrrc23 Δ/Δ sperm specifically. ( C ) Overwrapped STA images from 96 nm-doublet repeats from WT (gray) and Lrrc23 Δ/Δ (gold) sperm, and Chlamydomonas reinhardtii (cyan). ( D ) A proposed model of impaired sperm motility and male infertility by the LRRC23 loss of function.

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques:

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: LRRC23 mutation disrupts the third radial spoke (RS) in sperm flagellum. ( A ) Immunostaining of flagellar proteins in different compartments. Shown are midpiece (TOM20), annulus (SEPT4 and SEPT12), fibrous sheath (AKAP4), outer dense fiber (ODF2), and axoneme (acetylated tubulin, AcTub) in Lrrc23 +/Δ ( top ) and Lrrc23 Δ/Δ ( bottom ) sperm. Magnified insets are represented for annulus proteins (scale bars in insets = 2μm). Fluorescence and corresponding DIC images are merged. Sperm heads were counter stained with Hoechst. Experiments were performed with three biological replications. (B) Transmission electron microscopy images of Lrrc23 +/Δ ( left ) and Lrrc23 Δ/Δ ( right ) sperm. Shown are longitudinal section of sperm flagella. M, mitochondria; ODF, outer dense fiber; AX, axoneme; CP, central pair; MT, microtubule; FS, fibrous sheath. ( C ) Cryo-electron tomography (cryo-ET) of WT and Lrrc23 Δ/Δ sperm flagella. Shown are representative tomographic slices from WT ( left ) and Lrrc23 Δ/Δ sperm ( right ). The 9+2 axonemal structure are shown in both WT and Lrrc23 Δ/Δ in cross-sectional view ( left ). Axonemal structures are shown with proximal side of the flagellum on the left in longitudinal view ( right ). Magnified insets ( bottom ) reveal that RS1, 2, and 3 are shown in WT sperm ( left , filled arrowheads) but RS3, especially head part, is not clearly visible ( right , red arrowheads) in Lrrc23 Δ/Δ sperm. Lrrc23 +/Δ ( A and B ) or WT ( C ) sperm were used for positive control.

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Mutagenesis, Immunostaining, Fluorescence, Staining, Transmission Assay, Electron Microscopy, Tomography, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Schematic of the screening for candidate membrane proteins involved in SARS-CoV-2 entry. Schematic illustration of the labeling procedure according to EMARS. After EMARS reaction, the fluorescein-labeled proteins were purified and then analyzed using mass spectrometry.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Labeling, Purification, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: SARS-CoV-2 spike protein-based EMARS probes. ( A ) ACE2 expression in Caco-2 and A549 cells. Western blot analysis of Caco-2 and A549 cell lysates; 10 μg protein samples were subjected to SDS-PAGE (on 10% gels) and stained with anti-ACE2 antibody. Arrows indicate bands of the ACE2 protein. ( B ) Immunocytochemical staining of ACE2 in Caco-2 and A549 cells. Staining with the anti-ACE2 antibody (ACE2+2 nd 568) was performed as described in Experimental procedure . Negative control samples (2 nd 568) were also prepared simultaneously. White bar: 100 μm. ( C ) Immunocytochemical staining of SARS-CoV-2 spike proteins in Caco-2 and A549 cells. Staining of monovalent Alexa Fluor 488-labeled spike proteins (spike-488) and the two-step staining (spike protein followed by Alexa Fluor 488 secondary antibody; spike+2 nd 488) were performed with DIC images. Negative control samples (cAb-488 or 2 nd 488) were also prepared simultaneously. White bar: 100 μm.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Expressing, Western Blot, SDS Page, Staining, Negative Control, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Proximity labeling near the cell membrane-bound SARS-CoV-2 spike protein. ( A, B ) Fluorescein-labeled proximal proteins around cell membrane-bound SARS-CoV-2 spike proteins. The EMARS reaction described in the “Experimental procedure” was performed in Caco-2 ( A ) and A549 ( B ) cells using a spike protein ( Spike (RBD) ) and HRP-conjugated anti-mouse IgG ( mouse HRP ). The EMARS products were subsequently subjected to Western blot analysis to detect fluorescein-labeled proteins as candidate proximal proteins. In Caco-2 cells, HRP-conjugated Cholera Toxin B Subunit B ( CTxB-HRP ) was used for EMARS reaction as the positive control for membrane protein labeling. For loading controls, the PVDF membrane was stained with Coomassie Brilliant Blue after western blot analysis (right column)

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Labeling, Western Blot, Positive Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Co-localization of the identified proteins with cell membrane-bound SARS-CoV-2 spike proteins. Representative images of co-localization with SARS-CoV-2 spike proteins and the identified membrane proteins. Caco-2 cells were co-stained for SARS-CoV-2 spike protein (green) and the antibodies recognizing ACE2, CD133, Cadherin 17, DPP4, and VAPA (Red). The resulting specimens were subsequently stained with appropriate secondary antibodies and DAPI (Blue), then observed using confocal microscopy (20× objective). Co-localization is indicated in yellow in the “Merge” images. White bar: 10 μm.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Staining, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Candidate proteins located near SARS-CoV-2 spike proteins. ( A to D ) Morphological observation of SARS-CoV-2 spike proteins and the identified membrane proteins. Caco-2 cells observed using electron microscopy. Cultured Caco-2 cells were fixed and co-stained with the SARS-CoV-2 spike protein (indicated as 20 nm particles), and candidate molecules identified. CD133 ( A ), DPP4 ( B ), CDH17 ( C ), and VAPA ( D ) are indicated as 10 nm particles. Red arrows indicate the locations of SARS-CoV-2 spike proteins. Yellow arrow heads indicate the location of each candidate protein. Scale bar; 200 or 500 nm.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Electron Microscopy, Cell Culture, Staining

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: In vitro infection assay of SARS-CoV-2 pseudovirus. ( A ) Expression of ACE2 and candidate membrane proteins in transfectant HEK293 cells. Western blot analysis of transfectant cell lysates; Each cell lysates were subjected to SDS-PAGE (on 6 to 10% gels) and stained with antibodies recognizing ACE2 or candidate membrane proteins. Arrows indicate bands of the target proteins. The CBB staining image indicates load control. Asterisks indicate predicted nonspecific bands. ( B ) Schematic illustration of the assay procedure using HEK293T transfectant host cells. ( C ) Representative images of GFP-positive P-ACE2 cells after pSARS-CoV-2 infection. ACE2-expressing HEK293T cells were treated (pSARS-CoV-2 (+)) or not treated (pSARS-CoV-2 (-)) with pSARS-CoV-2, followed by fluorescein microscopic observation. Two independent experiments were carried out. White bar: 100 μm. ( D-F ) Flow cytometric analysis of pSARS-CoV-2-infected cells. P-ACE2 cells ( D ), candidate protein-single expressing cells ( E ), and candidate protein-coexpressing P-ACE2 cells ( F ) were analyzed using BD FACS Canto II. GFP-positive cells were defined as the infected cells with a GFP fluorescence intensity of 10 3 or higher (P3 area). Two ( E ) or five ( D and F ) independent replications were carried out in each experiment. ( G ) Increase in pSARS-CoV-2 infection in candidate protein-coexpressing P-ACE2 cells. The number of GFP-positive cells in each cell was quantified using flow cytometry. The number of infected cells (GFP-positive) in P-ACE2–CD133, –CDH17, and –VAPA was significantly higher than that in P-ACE2 cells ( P < 0.05 or P < 0.005; Dunnett's test), but not in P-ACE2-GPC3 (N.D.) as the negative control.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: In Vitro, Infection, Expressing, Transfection, Western Blot, SDS Page, Staining, Fluorescence, Flow Cytometry, Negative Control

Journal: Viruses

Article Title: Characterization of Immune Response towards Generation of Universal Anti-HA-Stalk Antibodies after Immunization of Broiler Hens with Triple H5N1/NA-HA-M1 VLPs

doi: 10.3390/v14040730

Figure Lengend Snippet: Production of H5N1 avian influenza triple H5N1/NA-HA-M1 VLPs. ( A ) Schematic representation of the tricistronic expression cassette. ( B ) Western blotting analysis of NA-HA-M1 VLPs purified on a 10%–60% sucrose gradient. Antibodies used for the detection of HA, NA and M1 proteins: mouse monoclonal anti-HA H5N1 antibodies, mouse monoclonal anti-M1 influenza antibodies and rabbit polyclonal anti-avian influenza A neuraminidase antibody. ( C ) Schematic representation of the predicted structure of triple H5N1/NA-HA-M1 VLPs. Structural proteins are color-coded according to the scheme of the expression cassette: NA—yellow, HA—blue, M1—green.

Article Snippet: Antibodies: Anti-H5N1 virus A/Ck/Scot/59 polyclonal chicken antibodies (cat. No RAA7002, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-H5N2 virus A/Ost/Den/72420/96 polyclonal chicken antibodies (cat. No RAA7003, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-M1 influenza mouse monoclonal (cat. No ab22396, Abcam Inc., Waltham, MA USA); anti-avian influenza A neuraminidase antibody (cat. No ab21304, Abcam Inc., Waltham, MA USA); anti-H1N1 mouse monoclonal antibodies (cat. No 11048-MM08, SinoBiological Inc., Beijing, China); FI6—highly specific humanized synthetic universal neutralizing monoclonal antibodies selected from plasma cells that bind group 1 and 2 influenza A HA (kind gift from Dr. Krzysztof Lacek and Alfredo Nicosia); anti-HA H5N1 mouse monoclonal antibodies (cat. No 11048-MM06, SinoBiological Inc., Beijing, China); anti-HA H3N2 mouse monoclonal antibodies (cat. No 11056-MM03, SinoBiological Inc., Beijing, China); anti-HA H7N9 mouse monoclonal antibodies (cat. No 11082-MM04, SinoBiological Inc., Beijing, China).

Techniques: Expressing, Western Blot, Purification

Journal: Viruses

Article Title: Characterization of Immune Response towards Generation of Universal Anti-HA-Stalk Antibodies after Immunization of Broiler Hens with Triple H5N1/NA-HA-M1 VLPs

doi: 10.3390/v14040730

Figure Lengend Snippet: Characterization of H5N1 avian influenza virus triple H5N1/NA-HA-M1 VLPs. ( A ) Transmission electron microscopy of triple H5N1/NA-HA-M1 VLPs and influenza A/H5N2 virus. Scale bar = 1 µm is shown in the right bottom corner of the images. ( B ) Hemagglutination assay. The AIV A/ostrich/Denmark/725/96 (H5N2) was used as a positive control. The HA titer was determined as the reciprocal of the highest dilution with HA activity. Each dot represents HA titers obtained in each experiment. The bars represent the median values of obtained HA titers. ( C ) Neuraminidase activity assay. The AIV A/Ck/Scot/59 (H5N1) was used as a positive control. For each assay, the mean value from three independent experiments performed is presented. The mean A560 values and standard deviations are shown on the y-axis.

Article Snippet: Antibodies: Anti-H5N1 virus A/Ck/Scot/59 polyclonal chicken antibodies (cat. No RAA7002, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-H5N2 virus A/Ost/Den/72420/96 polyclonal chicken antibodies (cat. No RAA7003, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-M1 influenza mouse monoclonal (cat. No ab22396, Abcam Inc., Waltham, MA USA); anti-avian influenza A neuraminidase antibody (cat. No ab21304, Abcam Inc., Waltham, MA USA); anti-H1N1 mouse monoclonal antibodies (cat. No 11048-MM08, SinoBiological Inc., Beijing, China); FI6—highly specific humanized synthetic universal neutralizing monoclonal antibodies selected from plasma cells that bind group 1 and 2 influenza A HA (kind gift from Dr. Krzysztof Lacek and Alfredo Nicosia); anti-HA H5N1 mouse monoclonal antibodies (cat. No 11048-MM06, SinoBiological Inc., Beijing, China); anti-HA H3N2 mouse monoclonal antibodies (cat. No 11056-MM03, SinoBiological Inc., Beijing, China); anti-HA H7N9 mouse monoclonal antibodies (cat. No 11082-MM04, SinoBiological Inc., Beijing, China).

Techniques: Virus, Transmission Assay, Electron Microscopy, Hemagglutination Assay, Positive Control, Activity Assay

Journal: Viruses

Article Title: Characterization of Immune Response towards Generation of Universal Anti-HA-Stalk Antibodies after Immunization of Broiler Hens with Triple H5N1/NA-HA-M1 VLPs

doi: 10.3390/v14040730

Figure Lengend Snippet: Schematic timeline of immunization of the broiler hens with triple H5N1/NA-HA-M1 VLPs. Red droplets represent the days of blood collection.

Article Snippet: Antibodies: Anti-H5N1 virus A/Ck/Scot/59 polyclonal chicken antibodies (cat. No RAA7002, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-H5N2 virus A/Ost/Den/72420/96 polyclonal chicken antibodies (cat. No RAA7003, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-M1 influenza mouse monoclonal (cat. No ab22396, Abcam Inc., Waltham, MA USA); anti-avian influenza A neuraminidase antibody (cat. No ab21304, Abcam Inc., Waltham, MA USA); anti-H1N1 mouse monoclonal antibodies (cat. No 11048-MM08, SinoBiological Inc., Beijing, China); FI6—highly specific humanized synthetic universal neutralizing monoclonal antibodies selected from plasma cells that bind group 1 and 2 influenza A HA (kind gift from Dr. Krzysztof Lacek and Alfredo Nicosia); anti-HA H5N1 mouse monoclonal antibodies (cat. No 11048-MM06, SinoBiological Inc., Beijing, China); anti-HA H3N2 mouse monoclonal antibodies (cat. No 11056-MM03, SinoBiological Inc., Beijing, China); anti-HA H7N9 mouse monoclonal antibodies (cat. No 11082-MM04, SinoBiological Inc., Beijing, China).

Techniques:

Journal: Viruses

Article Title: Characterization of Immune Response towards Generation of Universal Anti-HA-Stalk Antibodies after Immunization of Broiler Hens with Triple H5N1/NA-HA-M1 VLPs

doi: 10.3390/v14040730

Figure Lengend Snippet: End-point titration of chicken sera after immunization with triple H5N1/NA-HA-M1 VLPs. ELISA plates were coated with reference antigen inactivated H5N1 virus. Serial dilutions of chicken A/H5N1/HPAI polyclonal antibodies served as a positive control. Chicken serum from day 0 and control chicken serum served as a background. The dilution factor of the pooled sera is shown on the x-axis. For each ELISA, the mean value from three independent experiments performed is presented. The mean A450 values and standard deviations are shown on the y-axis.

Article Snippet: Antibodies: Anti-H5N1 virus A/Ck/Scot/59 polyclonal chicken antibodies (cat. No RAA7002, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-H5N2 virus A/Ost/Den/72420/96 polyclonal chicken antibodies (cat. No RAA7003, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-M1 influenza mouse monoclonal (cat. No ab22396, Abcam Inc., Waltham, MA USA); anti-avian influenza A neuraminidase antibody (cat. No ab21304, Abcam Inc., Waltham, MA USA); anti-H1N1 mouse monoclonal antibodies (cat. No 11048-MM08, SinoBiological Inc., Beijing, China); FI6—highly specific humanized synthetic universal neutralizing monoclonal antibodies selected from plasma cells that bind group 1 and 2 influenza A HA (kind gift from Dr. Krzysztof Lacek and Alfredo Nicosia); anti-HA H5N1 mouse monoclonal antibodies (cat. No 11048-MM06, SinoBiological Inc., Beijing, China); anti-HA H3N2 mouse monoclonal antibodies (cat. No 11056-MM03, SinoBiological Inc., Beijing, China); anti-HA H7N9 mouse monoclonal antibodies (cat. No 11082-MM04, SinoBiological Inc., Beijing, China).

Techniques: Titration, Enzyme-linked Immunosorbent Assay, Virus, Positive Control, Control

Journal: Viruses

Article Title: Characterization of Immune Response towards Generation of Universal Anti-HA-Stalk Antibodies after Immunization of Broiler Hens with Triple H5N1/NA-HA-M1 VLPs

doi: 10.3390/v14040730

Figure Lengend Snippet: Dynamics of anti-H5N1 IgY level in sera of immunized chickens. Kinetics of antibody titer in chickens ( n = 5) following prime and boost vaccinations with triple H5N1/NA-HA-M1 VLPs were measured via ELISA test. The median (thick line) is shown with the interquartile 25% and 75% range (narrow lines). The day of the serum collection is shown on the x-axis. The A450 values are shown on the y-axis. Statistical analysis was performed using the nonparametric Kruskal–Wallis test ( p = 0.05) and Benjamini, Krieger and Yekutieli multiple comparison test ( p = 0.05). Statistical differences were detected between days 0–41 and 0–34 ( p = 0.014) and shown on the graph as a star symbol.

Article Snippet: Antibodies: Anti-H5N1 virus A/Ck/Scot/59 polyclonal chicken antibodies (cat. No RAA7002, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-H5N2 virus A/Ost/Den/72420/96 polyclonal chicken antibodies (cat. No RAA7003, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-M1 influenza mouse monoclonal (cat. No ab22396, Abcam Inc., Waltham, MA USA); anti-avian influenza A neuraminidase antibody (cat. No ab21304, Abcam Inc., Waltham, MA USA); anti-H1N1 mouse monoclonal antibodies (cat. No 11048-MM08, SinoBiological Inc., Beijing, China); FI6—highly specific humanized synthetic universal neutralizing monoclonal antibodies selected from plasma cells that bind group 1 and 2 influenza A HA (kind gift from Dr. Krzysztof Lacek and Alfredo Nicosia); anti-HA H5N1 mouse monoclonal antibodies (cat. No 11048-MM06, SinoBiological Inc., Beijing, China); anti-HA H3N2 mouse monoclonal antibodies (cat. No 11056-MM03, SinoBiological Inc., Beijing, China); anti-HA H7N9 mouse monoclonal antibodies (cat. No 11082-MM04, SinoBiological Inc., Beijing, China).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: Viruses

Article Title: Characterization of Immune Response towards Generation of Universal Anti-HA-Stalk Antibodies after Immunization of Broiler Hens with Triple H5N1/NA-HA-M1 VLPs

doi: 10.3390/v14040730

Figure Lengend Snippet: HI titer of pooled chicken sera collected after immunization with triple H5N1/NA-HA-M1 VLPs. H5N1 A/Ck/Scot/59 and H5N2 A/Ost/Den/72420/96 antibodies were used as a positive control. Sera from chickens vaccinated with PBS/ICF mixture served as a negative control. HI assay was performed in triplicates.

Article Snippet: Antibodies: Anti-H5N1 virus A/Ck/Scot/59 polyclonal chicken antibodies (cat. No RAA7002, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-H5N2 virus A/Ost/Den/72420/96 polyclonal chicken antibodies (cat. No RAA7003, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-M1 influenza mouse monoclonal (cat. No ab22396, Abcam Inc., Waltham, MA USA); anti-avian influenza A neuraminidase antibody (cat. No ab21304, Abcam Inc., Waltham, MA USA); anti-H1N1 mouse monoclonal antibodies (cat. No 11048-MM08, SinoBiological Inc., Beijing, China); FI6—highly specific humanized synthetic universal neutralizing monoclonal antibodies selected from plasma cells that bind group 1 and 2 influenza A HA (kind gift from Dr. Krzysztof Lacek and Alfredo Nicosia); anti-HA H5N1 mouse monoclonal antibodies (cat. No 11048-MM06, SinoBiological Inc., Beijing, China); anti-HA H3N2 mouse monoclonal antibodies (cat. No 11056-MM03, SinoBiological Inc., Beijing, China); anti-HA H7N9 mouse monoclonal antibodies (cat. No 11082-MM04, SinoBiological Inc., Beijing, China).

Techniques: Positive Control, Negative Control, HI Assay

Journal: Viruses

Article Title: Characterization of Immune Response towards Generation of Universal Anti-HA-Stalk Antibodies after Immunization of Broiler Hens with Triple H5N1/NA-HA-M1 VLPs

doi: 10.3390/v14040730

Figure Lengend Snippet: Construction and characterization of HA-stalk universal influenza antigen from homologous H5N1 HPAI virus strain. ( A ) Schematic representation of the full-length HA (top panel) and HA-stalk constructs (bottom panel). To obtain the HA-stalk construct, a glycine linker was added between cysteines in positions C52 and C277, replacing the head region of the HA1 domain. ( B ) Schematic representation of the predicted structure of HA monomer and HA-stalk monomer. ( C ) Expression of H5N1 HA-stalk in insect cells was confirmed by IPMA with anti-H5N1 monoclonal antibodies. Full-length HA from the H5N1 strain was used as a positive control. Cells infected with the wild type baculovirus were used as a negative control. Images were taken at ×10 magnification. ( D ) Reactivity of the HA-stalk antigen and full-length HA from H5N1 in the IPMA with broadly neutralizing universal FI6 human antibodies. HA-stalk and full-length HA from the H5N1 strain was detected in transfected insect cells. Images were taken at ×20 magnification.

Article Snippet: Antibodies: Anti-H5N1 virus A/Ck/Scot/59 polyclonal chicken antibodies (cat. No RAA7002, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-H5N2 virus A/Ost/Den/72420/96 polyclonal chicken antibodies (cat. No RAA7003, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-M1 influenza mouse monoclonal (cat. No ab22396, Abcam Inc., Waltham, MA USA); anti-avian influenza A neuraminidase antibody (cat. No ab21304, Abcam Inc., Waltham, MA USA); anti-H1N1 mouse monoclonal antibodies (cat. No 11048-MM08, SinoBiological Inc., Beijing, China); FI6—highly specific humanized synthetic universal neutralizing monoclonal antibodies selected from plasma cells that bind group 1 and 2 influenza A HA (kind gift from Dr. Krzysztof Lacek and Alfredo Nicosia); anti-HA H5N1 mouse monoclonal antibodies (cat. No 11048-MM06, SinoBiological Inc., Beijing, China); anti-HA H3N2 mouse monoclonal antibodies (cat. No 11056-MM03, SinoBiological Inc., Beijing, China); anti-HA H7N9 mouse monoclonal antibodies (cat. No 11082-MM04, SinoBiological Inc., Beijing, China).

Techniques: Virus, Construct, Expressing, Positive Control, Infection, Negative Control, Transfection

Journal: Viruses

Article Title: Characterization of Immune Response towards Generation of Universal Anti-HA-Stalk Antibodies after Immunization of Broiler Hens with Triple H5N1/NA-HA-M1 VLPs

doi: 10.3390/v14040730

Figure Lengend Snippet: Expression of HA-stalk antigens from the 1 and 2 HA groups in mammalian cells. HEK293 cells were transfected with HA-stalk H1N1, H5N1 and H7N9 constructs. Protein expression was detected using different monoclonal antibodies specific for H1N1, H5N1 and H7N9 influenza strains. Non-transfected cells were used as a negative control. Images were taken at ×40 magnification.

Article Snippet: Antibodies: Anti-H5N1 virus A/Ck/Scot/59 polyclonal chicken antibodies (cat. No RAA7002, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-H5N2 virus A/Ost/Den/72420/96 polyclonal chicken antibodies (cat. No RAA7003, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-M1 influenza mouse monoclonal (cat. No ab22396, Abcam Inc., Waltham, MA USA); anti-avian influenza A neuraminidase antibody (cat. No ab21304, Abcam Inc., Waltham, MA USA); anti-H1N1 mouse monoclonal antibodies (cat. No 11048-MM08, SinoBiological Inc., Beijing, China); FI6—highly specific humanized synthetic universal neutralizing monoclonal antibodies selected from plasma cells that bind group 1 and 2 influenza A HA (kind gift from Dr. Krzysztof Lacek and Alfredo Nicosia); anti-HA H5N1 mouse monoclonal antibodies (cat. No 11048-MM06, SinoBiological Inc., Beijing, China); anti-HA H3N2 mouse monoclonal antibodies (cat. No 11056-MM03, SinoBiological Inc., Beijing, China); anti-HA H7N9 mouse monoclonal antibodies (cat. No 11082-MM04, SinoBiological Inc., Beijing, China).

Techniques: Expressing, Transfection, Construct, Negative Control

Journal: Viruses

Article Title: Characterization of Immune Response towards Generation of Universal Anti-HA-Stalk Antibodies after Immunization of Broiler Hens with Triple H5N1/NA-HA-M1 VLPs

doi: 10.3390/v14040730

Figure Lengend Snippet: Cross-reactivity of chicken sera obtained after vaccination with triple H5N1/NA-HA-M1 VLPs with the HA-stalk antigens from the 1 and 2 HA groups. Detection of HA-stalk from H1N1, H5N1 and H7N9 strains was performed on transfected HEK293 cells. Sera from unvaccinated hens were used as a background. Non-transfected cells were used as a negative control. Images were taken at ×40 magnification.

Article Snippet: Antibodies: Anti-H5N1 virus A/Ck/Scot/59 polyclonal chicken antibodies (cat. No RAA7002, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-H5N2 virus A/Ost/Den/72420/96 polyclonal chicken antibodies (cat. No RAA7003, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-M1 influenza mouse monoclonal (cat. No ab22396, Abcam Inc., Waltham, MA USA); anti-avian influenza A neuraminidase antibody (cat. No ab21304, Abcam Inc., Waltham, MA USA); anti-H1N1 mouse monoclonal antibodies (cat. No 11048-MM08, SinoBiological Inc., Beijing, China); FI6—highly specific humanized synthetic universal neutralizing monoclonal antibodies selected from plasma cells that bind group 1 and 2 influenza A HA (kind gift from Dr. Krzysztof Lacek and Alfredo Nicosia); anti-HA H5N1 mouse monoclonal antibodies (cat. No 11048-MM06, SinoBiological Inc., Beijing, China); anti-HA H3N2 mouse monoclonal antibodies (cat. No 11056-MM03, SinoBiological Inc., Beijing, China); anti-HA H7N9 mouse monoclonal antibodies (cat. No 11082-MM04, SinoBiological Inc., Beijing, China).

Techniques: Transfection, Negative Control

Journal: Viruses

Article Title: Characterization of Immune Response towards Generation of Universal Anti-HA-Stalk Antibodies after Immunization of Broiler Hens with Triple H5N1/NA-HA-M1 VLPs

doi: 10.3390/v14040730

Figure Lengend Snippet: Alignment of amino acid sequences coding LAH regions from H5N1, pH1N1, H7N9 and H3N2 HA protein. ( A ) Alignment view with consensus sequence where the highest similarity is shown as a green colour. ( B ) Matrix showing the percentage of sequence identity between sequences.

Article Snippet: Antibodies: Anti-H5N1 virus A/Ck/Scot/59 polyclonal chicken antibodies (cat. No RAA7002, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-H5N2 virus A/Ost/Den/72420/96 polyclonal chicken antibodies (cat. No RAA7003, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-M1 influenza mouse monoclonal (cat. No ab22396, Abcam Inc., Waltham, MA USA); anti-avian influenza A neuraminidase antibody (cat. No ab21304, Abcam Inc., Waltham, MA USA); anti-H1N1 mouse monoclonal antibodies (cat. No 11048-MM08, SinoBiological Inc., Beijing, China); FI6—highly specific humanized synthetic universal neutralizing monoclonal antibodies selected from plasma cells that bind group 1 and 2 influenza A HA (kind gift from Dr. Krzysztof Lacek and Alfredo Nicosia); anti-HA H5N1 mouse monoclonal antibodies (cat. No 11048-MM06, SinoBiological Inc., Beijing, China); anti-HA H3N2 mouse monoclonal antibodies (cat. No 11056-MM03, SinoBiological Inc., Beijing, China); anti-HA H7N9 mouse monoclonal antibodies (cat. No 11082-MM04, SinoBiological Inc., Beijing, China).

Techniques: Sequencing

Journal: Viruses

Article Title: Characterization of Immune Response towards Generation of Universal Anti-HA-Stalk Antibodies after Immunization of Broiler Hens with Triple H5N1/NA-HA-M1 VLPs

doi: 10.3390/v14040730

Figure Lengend Snippet: Cross-reactivity of chicken sera obtained after vaccination with triple H5N1/NA-HA-M1 VLPs with the LAH peptide from H3 from 2 HA group. The antibody titer in chickens ( n = 5) before (gray) and after (black) vaccination with triple H5N1/NA-HA-M1 VLPs was measured via the peptide ELISA test. The mean OD values and standard deviations are shown on the y-axis. The tested chickens are shown on the x-axis. Statistical analysis was performed using a nonparametric Wilcoxon test ( p = 0.05) for paired groups.

Article Snippet: Antibodies: Anti-H5N1 virus A/Ck/Scot/59 polyclonal chicken antibodies (cat. No RAA7002, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-H5N2 virus A/Ost/Den/72420/96 polyclonal chicken antibodies (cat. No RAA7003, Animal Health and Veterinary Laboratories Agency, Weybridge, UK); anti-M1 influenza mouse monoclonal (cat. No ab22396, Abcam Inc., Waltham, MA USA); anti-avian influenza A neuraminidase antibody (cat. No ab21304, Abcam Inc., Waltham, MA USA); anti-H1N1 mouse monoclonal antibodies (cat. No 11048-MM08, SinoBiological Inc., Beijing, China); FI6—highly specific humanized synthetic universal neutralizing monoclonal antibodies selected from plasma cells that bind group 1 and 2 influenza A HA (kind gift from Dr. Krzysztof Lacek and Alfredo Nicosia); anti-HA H5N1 mouse monoclonal antibodies (cat. No 11048-MM06, SinoBiological Inc., Beijing, China); anti-HA H3N2 mouse monoclonal antibodies (cat. No 11056-MM03, SinoBiological Inc., Beijing, China); anti-HA H7N9 mouse monoclonal antibodies (cat. No 11082-MM04, SinoBiological Inc., Beijing, China).

Techniques: Peptide ELISA

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RNAi screening identifies RABGAP1L as an IAV restriction factor (A) Schematic representation of recombinant IAV WSN/33 in which the coding region for the hemagglutinin (HA) glycoprotein has been replaced by Renilla luciferase (WSN/33- Renilla ). (B) RNAi-screening experimental workflow. (C) MRC-5-HA cells were transfected for 30 h with individual siRNAs targeting MX1 or IFITM3 or with a non-targeting (NT) control siRNA. Following stimulation with IFNα2 (1,000 U/mL or mock) for 16 h, cells were infected with WSN/33- Renilla (MOI 5 PFU/cell) in the presence of the live-cell substrate EnduRen. Luciferase activity was monitored up to 12 h post-infection (p.i.), and the area under the curve (AUC) was calculated as indicated. Mean values from 50 technical replicates across two independent biological experiments are plotted, with error bars representing SDs. (D) Hit criteria for RNAi screening. In a primary screen following the workflow in (B), 100 putative ISGs were silenced with four individual siRNAs each. Twenty-two genes met the threshold, and 20 were re-tested in a confirmation screen. Applying the same hit criteria, a total of 8 putative ISGs were confirmed in both screening rounds. (E) Heatmap showing Z scores of positive controls ( MX1 and IFITM3 ) and the top 8 hits from the two RNAi-screening rounds. Columns represent individual siRNAs targeting genes listed in rows. See also .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Recombinant, Luciferase, Transfection, Infection, Activity Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: IFN-mediated restriction of IAV by RABGAP1L (A) A549 cells were transfected with the indicated siRNAs for 32 or 60 h prior to lysis and assessment of cell viability using CellTiter-Glo. An NT siRNA and an siRNA targeting IRF9 were used as negative controls. siRPS is an siRNA targeting the essential gene RPS27A and thus acted as a positive control for cell toxicity. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (B and C) A549 cells were transfected with the indicated siRNAs 30 h prior to IFNα2 treatment (1,000 U/mL or mock). Sixteen hours post-IFN stimulation, cells were infected with WSN/33- Renilla (MOI 1 PFU/cell), and luciferase activity was monitored every 2 h for a total of 12 h. The NT siRNA and siRNA targeting IRF9 were used as controls. (C) The AUC was calculated from measured relative light units (RLUs) over time. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (D) In parallel to (B) and (C), cells were harvested for western blot analysis 16 h post-IFN stimulation. Proteins of interest were detected as indicated. RABGAP1L (RG1L) isoforms corresponding to detected bands are highlighted. (E) Schematic representation of RABGAP1L isoforms A, G, H, and I, showing the phosphotyrosine-binding (PTB) domain, the kinesin-like (kin) domain, and the Tre-2/Bub2/Cdc16 (TBC) domain. Isoform G further contains a domain of unknown function (DUF3084). (F) Immunofluorescence analysis of A549 cells stably expressing either empty vector (EV) or RABGAP1L isoforms A, G, H, and I. Cells were fixed and stained for RABGAP1L (red); nuclei were stained with DAPI (blue). Scale bar represents 25 μm. Representative confocal-microscopy images from at least two biologically independent experiments are shown. (G) Cells described in (F) were harvested for western-blot analysis. Proteins of interest were detected with the indicated antibodies. Images are representative of three biologically independent experiments. (H) Cells described in (F) and (G) were treated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with WSN/33 (MOI 0.001 PFU/cell). Supernatants were collected 48 h p.i. and titrated on Madin-Darby canine kidney (MDCK) cells to determine viral titers. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance in (C) and (H) was determined using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗∗ p < 0.0001; ns, non-significant). See also and .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Transfection, Lysis, Positive Control, Infection, Luciferase, Activity Assay, Western Blot, Binding Assay, Immunofluorescence, Stable Transfection, Expressing, Plasmid Preparation, Staining, Confocal Microscopy, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RABGAP1L overexpression restricts selected positive- and negative-sense RNA viruses (A) A549 cells stably expressing GFP or RABGAP1L (RG1L) were stimulated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with different Renilla luciferase-encoding IAVs: H1N1 (WSN/33, MOI 1 PFU/cell), pdmH1N1 (Neth/09, MOI 5 PFU/cell), or H5N1 (Viet/04, MOI 0.5 PFU/cell). EnduRen live-cell substrate was added p.i., and the luciferase activity was monitored every 2 h for a total of 11 h. The AUC was calculated from RLUs up to 11 h p.i. (B) Huh-7 cells stably expressing GFP or RG1L were treated as described in (A) and infected with WSN/33- Renilla (MOI 1 PFU/cell) or HCoV-229E- Renilla (MOI 5 PFU/cell). EnduRen was supplemented, and the luciferase activity was measured every 2 h for a total of 11 h. RLUs were used to calculate the AUC. (C–E) A549 cells expressing EV or RG1L were stimulated with IFNα2 (10, 100 or 1,000 U/mL or mock) for 4 h prior to infection with VSV-GFP (MOI 1 PFU/cell) (C) or for 16 h prior to infection with SeV-GFP (MOI ∼1 PFU/cell) (D) and NDV-GFP (MOI 1 PFU/cell) (E). GFP intensity was measured every 2 h for up to 72 h. The AUC was calculated from total green integrated intensity. (F and H) Calu-3 (F) or Vero-CCL81 (H) cells stably expressing GFP or RG1L were treated with IFNα2 (10, 100, or 1,000 U/mL or mock) for 16 h, followed by infection with WSN/33- Renilla (MOI 1 PFU/cell). EnduRen was added p.i., and the luciferase activity was monitored every 2 h for a total of 11 h. The AUC was calculated from RLUs. (G and I) Calu-3 (G) or Vero-CCL81 (I) cells stably expressing GFP or RG1L were treated as described in (F) prior to infection with SARS-CoV-2 (MOI 0.1 PFU/cell). Supernatants were collected 24 h p.i., and viral titers were determined by plaque assay in Vero-E6 cells. (A–I) Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance was determined comparing GFP-overexpressing with RG1L-overexpressing cells in equal treatment conditions in all panels using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗ p < 0.0002, ∗∗∗∗ p < 0.0001; ns, non-significant).

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Over Expression, Stable Transfection, Expressing, Infection, Luciferase, Activity Assay, Plaque Assay, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: The antiviral function of RABGAP1L relies on its catalytically active TBC domain and residues implicated in endosomal trafficking (A) Schematic representation of RG1L WT and the 421 mutant (RG1L 421) which lacks the C-terminal region downstream of the kin domain. (B) Immunofluorescence analysis of A549 cells stably expressing EV, RG1L WT, or RG1L 421. Cells were fixed and stained with the indicated antibodies. Scale bar represents 25 μm. (C) A549 cells stably expressing GFP, RG1L WT, or RG1L 421 were stimulated with IFNα2 (1,000 U/mL or mock) for 16 h prior to infection with WSN/33 (MOI 0.001 PFU/cell). Supernatants were collected after 48 h and titrated on MDCK cells. (D) Schematic representation of the TBC domain of RABGAP1L and the localization of mutants R584A (R mut ), Q621A (Q mut ), R584A-Q621A (RQ mut ), and KK784EE (KK mut ). KK mut has previously been shown to prevent interaction with the AnkB death domain (DD). (E) Western blot validation of RABGAP1L expression in A549 cells stably expressing RG1L WT or the indicated mutants. (F) Immunofluorescence analysis of cells described in (E) (here, EV was used as a control), fixed and stained with the indicated antibodies. Scale bar represents 25 μm. (G) Cells described in (E) were infected with WSN/33- Renilla (MOI 1 PFU/cell) following treatment with IFNα2 (1,000 U/mL or mock) for 16 h. The AUC was calculated from RLU values taken up to 11 h p.i. For (B), (E), and (F), representative data from three biologically independent experiments are shown. For (C) and (G), mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. Statistical significance was determined using one-way ANOVA following log transformation ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗∗ p < 0.0001). See also Figure S3 .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Mutagenesis, Immunofluorescence, Stable Transfection, Expressing, Staining, Infection, Western Blot, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: Proximity-labeling-based proteomics identifies the RABGAP1L host interactome (A) Schematic representation of TurboID-V5-tagged (T-V5) GFP (negative control) carrying a nuclear-export sequence (NES) or T-V5-tagged RABGAP1L (T-V5-RG1L). (B) Constructs described in (A) were stably expressed in A549 cells, and their expression was validated by immunofluorescence using an α-V5 (red) antibody. Nuclei were stained with DAPI (blue). Scale bar represents 25 μm. (C) Western blot analysis of cells described in (B) compared with A549 cells stably expressing untagged GFP or RABGAP1L (RG1L). Proteins of interest were detected with the indicated antibodies. (D) Cells described in (C) were stimulated with IFNα2 (1,000 U/mL or mock) 16 h prior to infection with WSN/33- Renilla (MOI 1 PFU/cell). The AUC was calculated from RLU values taken up to 11 h p.i. Mean values from three biologically independent experiments are plotted, with error bars representing SDs. Individual data points are shown. (E) Workflow of the TurboID proximity-labeling approach. Cells described in (B) were treated with IFNα2 (1,000 U/mL or mock) for 16 h, followed by treatment with biotin (500 μM) for 15 min. Following streptavidin-based affinity purification, peptides were generated and subjected to mass-spectrometry analyses. (F) Interactors specific to RABGAP1L (as compared to GFP-NES) identified using the protocol described in (E). Hits are listed with their gene names and sorted according to previously described functions. Most hits were identified in non-IFNα2-treated samples. Hits marked with an asterisk ( ∗ ) were identified in the presence and absence of IFNα2, and hits marked in bold were only identified in IFNα2-treated samples. (G) A549 cells stably expressing constructs introduced in (A) or T-V5-tagged RABGAP1L KK mut and RQ mut were subjected to the proximity labeling approach outlined in (E). Following streptavidin-based affinity purification (samples termed “eluates”), total lysates and eluates were analyzed by western blot. Proteins were detected with the indicated antibodies. Data obtained in (B), (C), and (G) are representative of three biologically independent experiments. For (D), statistical significance was determined using one-way ANOVA following log transformation (ns, non-significant). See also and Figure S4 .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Labeling, Negative Control, Sequencing, Construct, Stable Transfection, Expressing, Immunofluorescence, Staining, Western Blot, Infection, Affinity Purification, Generated, Mass Spectrometry, Transformation Assay

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet: RABGAP1L expression impacts host endosomal function and IAV uptake (A–C) A549 cells stably expressing RABGAP1L WT, the 421-truncation mutant, or EV were infected with WSN/33 (MOI 5 PFU/cell) for 1 h on ice. Three hours after incubation at 37°C, cells were fixed and stained with antibodies against RABGAP1L (red) and NP (green) (A). Nuclei were stained with DAPI (blue). Scale bar represents 25 μm. (B and C) Green mean fluorescent intensities (MFIs) of nuclear NP signals were quantified from fluorescent-microscopy images from (A) using ImageJ software. Individual cells are represented by single dots (B). Mean values of data from three biologically independent experiments in (B), normalized to EV, are shown in (C). (D) MDCK cells, expressing the constructs described in (A), were infected for 4 h at 37°C with WSN/33-pseudotyped β-lactamase-matrix protein (BlaM1) fusion protein VLPs prior to quantification of entry-positive cells via flow cytometry. Data represent means, with error bars showing SDs, from three biologically independent experiments. Individual data points are shown. (E) Experimental setup for immunofluorescence-based confocal microscopy to track early stages during IAV entry. Following infection with WSN/33 (MOI 25 PFU/cell or mock) for 1 h at 4°C, cells were fixed at the indicated timepoints. (F) A549 cells stably expressing RABGAP1L (RG1L) or EV were subjected to the experimental setup described in (E). The MFI of HA signals (green) at 0 min p.i. were quantified from confocal-microscopy images shown in Figure S6 A using ImageJ. Individual cells are represented by single dots. (G) Quantification of co-localizations between EEA1 and HA from confocal images shown in (H) and Figure S6 A using Imaris. Individual cells are represented by single dots. (H) Immunofluorescence analysis of RABGAP1L or EV-expressing A549 cells treated as described in (E). Cells were stained for early endosomes (EEA1, magenta), viral proteins (HA, green), and nuclei (DAPI, blue). Scale bar represents 25 μm. White arrows indicate co-localizations between EEA1 and HA. Representative images of at least nine analyzed cells per time point from at least two biologically independent experiments. (I) Quantification of co-localizations between EEA1 and HA from confocal images shown in Figure S6 B. Individual cells are represented by single dots. (J and K) Cells described in (A) were serum starved for 2 h prior to treatment with Dynasore (Dyn.; 100 μm) or DMSO for 1 h at 37°C. Cells were then incubated with Alexa-Fluor-488-conjugated transferrin (Tf-488) for 1 h at 4°C followed by a 10-min incubation at 37°C prior to fixation. (J) MFI quantification of Tf-488 signals from confocal-microscopy images shown in (K) using ImageJ software. Individual cells are represented by single dots. (K) Cells were stained with anti-transferrin receptor (TfR) antibody (magenta) and DAPI (blue) prior to analysis by confocal microscopy. Scale bar represents 25 μm. Representative images of at least 25 analyzed cells from two biologically independent experiments. Statistical significance was determined using unpaired nonparametric t test (B, F, G, and J), unpaired one-way ANOVA (C and D), or ordinary two-way ANOVA (I) ( ∗ p < 0.05, ∗∗ p < 0.002, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, non-significant). See also .

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Expressing, Stable Transfection, Mutagenesis, Infection, Incubation, Staining, Microscopy, Software, Construct, Flow Cytometry, Immunofluorescence, Confocal Microscopy

Journal: Cell Reports

Article Title: Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

doi: 10.1016/j.celrep.2022.110549

Figure Lengend Snippet:

Article Snippet: Proteins were detected by western blotting using the following primary antibodies: actin (rabbit, catalog no. A2103; Sigma-Aldrich), β-actin (mouse, catalog no.sc-47778; Santa Cruz), RABGAP1L (rabbit, catalog no. 13894-1-AP; proteintech), MxA (mouse ab143, kindly provided by Jovan Pavlovic, University of Zurich) , STAT1 (mouse, catalog no. sc-417; Santa Cruz), pSTAT1-Y701 (rabbit, catalog no. 7649S; Cell Signaling), IFI44 (rabbit, catalog no. HPA043858; Atlas Antibodies), FLAG M2 (mouse, catalog no. F1804; Sigma-Aldrich), PB1 (rabbit, catalog no. GTX125923; Genetex), PB2 (rabbit, inhouse), PA (rabbit, catalog no. GTX118991; Genetex), NP (mouse HB65, catalog no. H16-L10-4R5, ATCC), V5 (mouse, catalog no. MCA1360; Bio-Rad), VPS33A (rabbit, catalog no. 16896-1-AP, proteintech), RAB27B (rabbit, catalog no. 13412-1-AP, proteintech), SNF8 (mouse, catalog no. sc-390747, Santa Cruz), A/WSN/33 HA1 (rabbit, catalog no. 11692-T54; Sino Biological) and EEA1 (rabbit, catalog no. 2411, Cell Signaling).

Techniques: Recombinant, Transfection, Protease Inhibitor, Magnetic Beads, Electron Microscopy, Cell Viability Assay, Mutagenesis, Clone Assay, Luciferase, Staining, Labeling, Software, Real-time Polymerase Chain Reaction, Imaging, Laser-Scanning Microscopy, Microscopy

Journal: Frontiers in Endocrinology

Article Title: Integration of Metabolomics and Proteomics in Exploring the Endothelial Dysfunction Mechanism Induced by Serum Exosomes From Diabetic Retinopathy and Diabetic Nephropathy Patients

doi: 10.3389/fendo.2022.830466

Figure Lengend Snippet: Serum exosomes from diabetic retinopathy and nephropathy patients could induce endothelial dysfunction. (A) The typical pathological kidney images and fundus images obtained from healthy people and a diabetic microvascular disease patient. PAS : The DMC biopsy sample showed proliferation and swelling of the endothelial cell (blue arrow), reduplication (double contour appearance) of the glomerular basement membrane (red arrow), mesangial expansion (black arrow), arteriolar hyalinosis (green arrow). Scale bar: 50 μm. Methenamine silver: Renal biopsy samples from the DMC patient show the swelling of the endothelial cells (red arrow) and reduplication (double contour appearance) of the glomerular basement membrane (black arrow). Scale bar: 50 μm. Electron microscopy: The slice from the DMC patient showed mesangial expansion (red arrow). Scale bar: 5 μm. Fundus: The fundus from DMC showed microhemangioma (green arrow), retinal exudates (red arrow), intraretinal hemorrhage (black arrow), and intraretinal microvascular abnormalities (IRMAs; violet arrow). (B) Identification of serum exosomes from CON patients and DRDN patients by transmission electron microscopy (TEM). Scale bar: 200 nm. (C) Western blotting was used to look for the exosomal markers CD9, CD63, and TSG101 in exosome samples. (D) Analysis of the size distribution of exosomes from patients using the NanoSight technology; the average size of serum exosomes was 107.5 ± 55.2 nm. (E) Exosome tracing experiment captured by confocal microscope. Blue for DNA dyed by DAPI and green for exosomes derived from DR+DN patients dyed by PKH67. HGECs were subjected to 6, 12, and 24 h of incubation with exosomes. Scale bar: 25 μm. PAS, Periodic acid–Schiff stain; DMC, diabetic microvascular complications; CON, healthy controls; DRDN, patients diagnosed with both diabetic retinopathy and diabetic nephropathy.

Article Snippet: The primary antibodies were anti-CD9 (Abcam, Hong Kong, China; # ab92726), anti-CD63 (Abcam, ab216130), anti-TSG101 (Sino Biological, Beijing, China; #102286-T38), anti-CD31 (Zen-Bioscience, Chengdu, China; #383815), anti-von Willebrand factor (vWF), (Abcam, #ab154193), anti-intercellular adhesion molecule 1 (ICAM-1) (Abcam, #ab171123), anti-vascular cell adhesion protein 1 (VCAM-1) (Abcam, #ab134047), and anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (Goodhere, Hangzhou, China; #AB-P-R 001).

Techniques: Membrane, Electron Microscopy, Transmission Assay, Western Blot, Microscopy, Derivative Assay, Incubation, Staining

Journal: Nephron. Clinical Practice

Article Title: The COVID-Kidney Controversy: Can SARS-CoV-2 Cause Direct Renal Infection?

doi: 10.1159/000513789

Figure Lengend Snippet: Summary of studies as of November 30, 2020, that have investigated direct kidney infection by SARS-CoV-2

Article Snippet: Kudose et al. [ ]. J Am Soc Nephrol. USA , 17 (14 native kidneys and3 transplant allografts) , Kidney biopsy , RNA-ISH TEM IHC 1. Anti-SARS-NP antibody from clone ID: 001, rabbit, Sino Biological 2. Anti-SARS-S2 antibody from clone ID: 1A9, GTX632604, mouse, GeneTex , No SARS-CoV-2 detected.

Techniques: Infection, Staining

Journal: Cell death & disease

Article Title: TRIB3 promotes malignancy of head and neck squamous cell carcinoma via inhibiting ferroptosis.

doi: 10.1038/s41419-024-06472-5

Figure Lengend Snippet: Fig. 1 Associations between prognostic significance and TRIB3 expression in HNSCC. A A Heat Map of TRIB1, TRIB2, and TRIB3 gene expression in HNSCC and normal tissues. B, C Comparison analysis of TRIB3 levels among HNSCC tissues and normal tissues or adjacent tissues in the TCGA database or the CPTAC database. D ROC curve indicating the predictive value of TRIB3. E Comparison of the low and high expression of TRIB3 via Kaplan–Meier OS curve and survival status in GEO database or F TCGA database. G Comparison between the low and high expression of TRIB3 via Kaplan–Meier PFS curve and survival status in TCGA database. ***P < 0.001, **P < 0.01, *P < 0.05.

Article Snippet: Following are the antibodies utilized for immunofluorescence and their concentrations: TRIB3 (Proteintech, China, 13300-1-AP, 1:100), β-catenin (Cell Signaling Technology, 2677 S, 1:200), TCF4 (NOVUS, H00006925-M03, 10 μg/ml), Anti-rat IgG (abcam, ab15007, 1:1,000), Anti-mouse IgG (abcam, ab150115, 1:1,000).

Techniques: Expressing, Gene Expression, Comparison

Journal: Cell death & disease

Article Title: TRIB3 promotes malignancy of head and neck squamous cell carcinoma via inhibiting ferroptosis.

doi: 10.1038/s41419-024-06472-5

Figure Lengend Snippet: Fig. 2 TRIB3 silencing inhibits HNSCC cell viability and proliferation. A Western blot to detect protein levels of TRIB3 in normal NOK cell and HNSCC cell lines. Cal27 and FaDu cells were transfected with two independent TRIB3 shRNAs. Western blot (B) and qPCR (C) to detect TRIB3 levels in TRIB3-knockdown and control cells. D OD450 readings were plotted over time using the CCK8 assay. E, F Findings of a clonogenic analysis. Red circles demonstrated the counted clones. G Visualizztion of DNA replication using EdU. Red-stained cell nuclei demonstrated DNA replication. Scale bar: 50 μm. ***P < 0.001, **P < 0.01, *P < 0.05.

Article Snippet: Following are the antibodies utilized for immunofluorescence and their concentrations: TRIB3 (Proteintech, China, 13300-1-AP, 1:100), β-catenin (Cell Signaling Technology, 2677 S, 1:200), TCF4 (NOVUS, H00006925-M03, 10 μg/ml), Anti-rat IgG (abcam, ab15007, 1:1,000), Anti-mouse IgG (abcam, ab150115, 1:1,000).

Techniques: Western Blot, Transfection, Knockdown, Control, CCK-8 Assay, Clone Assay, Staining

Journal: Cell death & disease

Article Title: TRIB3 promotes malignancy of head and neck squamous cell carcinoma via inhibiting ferroptosis.

doi: 10.1038/s41419-024-06472-5

Figure Lengend Snippet: Fig. 3 Promotion of cell death by TRIB3 silencing largely through inhibiting ferroptosis. A GO analysis of DEGs between Cal27 TRIB3- knockdown and control cells. B CCK-8 assay detected the cell viability of Cal27 TRIB3-knockdown and control cells, treated with or without cell death inhibitors. C, D Lipid peroxidation of TRIB3-knockdown and control cells. E MDA in TRIB3-knockdown and control cells. F The Fe2+ level in control cells and TRIB3-knockdown. G Transmission electron microscopy images of Cal27 TRIB3-knockdown and control cells. The red arrows demonstrate mitochondria. Yellow arrows demonstrate autophagosomes. Scale bars: right, 500 nm; left, 1 µm. H qPCR analysis of CHAC1 expression in control and TRIB3-knockdown cells. ***P < 0.001, **P < 0.01, *P < 0.05.

Article Snippet: Following are the antibodies utilized for immunofluorescence and their concentrations: TRIB3 (Proteintech, China, 13300-1-AP, 1:100), β-catenin (Cell Signaling Technology, 2677 S, 1:200), TCF4 (NOVUS, H00006925-M03, 10 μg/ml), Anti-rat IgG (abcam, ab15007, 1:1,000), Anti-mouse IgG (abcam, ab150115, 1:1,000).

Techniques: Knockdown, Control, CCK-8 Assay, Transmission Assay, Electron Microscopy, Expressing

Journal: Cell death & disease

Article Title: TRIB3 promotes malignancy of head and neck squamous cell carcinoma via inhibiting ferroptosis.

doi: 10.1038/s41419-024-06472-5

Figure Lengend Snippet: Fig. 4 Induction of ferroptosis by TRIB3 silencing in HNSCC. A, B MDA in control and TRIB3-knockdown cells treated with Ferrostatin-1 (5 μm) or DFO (50 μm) for 48 h. C, D, E Lipid peroxidation in control and TRIB3-knockdown cells treated with Ferrostatin-1 (5 μm) or DFO (50 μm) for 48 h. F, G, H The level of Fe2+ in control and TRIB3-knockdown cells treated with Ferrostatin-1 (5 μm) or DFO (50 μm) for 48 h. I Schematic of TRIB3-knockdown subcutaneous tumors either treated with or without Liproxstatin-1. J, K Tumor growth and L weight in the xenograft model. M The expression of Ki67, TRIB3, 4-HNE using IHC assay. Scale bar: 100 μm. ***P < 0.001, **P < 0.01, *P < 0.05.

Article Snippet: Following are the antibodies utilized for immunofluorescence and their concentrations: TRIB3 (Proteintech, China, 13300-1-AP, 1:100), β-catenin (Cell Signaling Technology, 2677 S, 1:200), TCF4 (NOVUS, H00006925-M03, 10 μg/ml), Anti-rat IgG (abcam, ab15007, 1:1,000), Anti-mouse IgG (abcam, ab150115, 1:1,000).

Techniques: Control, Knockdown, Expressing

Journal: Cell death & disease

Article Title: TRIB3 promotes malignancy of head and neck squamous cell carcinoma via inhibiting ferroptosis.

doi: 10.1038/s41419-024-06472-5

Figure Lengend Snippet: Fig. 5 Interaction of TRIB3 with β-catenin and TCF4 to create a heterotrimeric complex. A KEGG analysis based on RNA-seq showed enrichment of DEGs in Wnt-signaling pathways. B Western blot to detect TCF4, β-catenin, and TRIB3 levels in the cytoplasm and nucleus of control and TRIB3-knockdown cells. C, D HNSCC cells extracts were immuno-precipitated and blotted with anti-TCF4, anti-TRIB3, or anti–β-catenin antibodies, respectively. Normal rabbit IgG was utilized as the control. E, F Co-IP of β-catenin and TCF4 in HNSCC cells with or without TRIB3 knockdown. ***P < 0.001, **P < 0.01, *P < 0.05.

Article Snippet: Following are the antibodies utilized for immunofluorescence and their concentrations: TRIB3 (Proteintech, China, 13300-1-AP, 1:100), β-catenin (Cell Signaling Technology, 2677 S, 1:200), TCF4 (NOVUS, H00006925-M03, 10 μg/ml), Anti-rat IgG (abcam, ab15007, 1:1,000), Anti-mouse IgG (abcam, ab150115, 1:1,000).

Techniques: RNA Sequencing, Protein-Protein interactions, Western Blot, Control, Knockdown, Co-Immunoprecipitation Assay

Journal: Cell death & disease

Article Title: TRIB3 promotes malignancy of head and neck squamous cell carcinoma via inhibiting ferroptosis.

doi: 10.1038/s41419-024-06472-5

Figure Lengend Snippet: Fig. 6 Inhibition of ALOXE3 activity by TRIB3‒β-catenin‒TCF4 heterotrimer complex. A Scatter plot of DEGs. B Overlay of downregulated genes (TRIB3-knockdown vs. control) with known suppressor genes in ferroptosis and upregulated genes (TRIB3-knockdown vs. control) with known driver genes in ferroptosis (up), respectively. Overlay of 12 down DEGs and 6 up DEGs with Cistrome Data Browser ChIP-seq (bottom). C, D qPCR to detect ALOXE3 and PARP8 levels in TRIB3 knockdown and control cells. E, F ChIP analyses of TCF4 binding on the ALOXE3 and PARP8 promoter in Cal27 TRIB3-overexpression cells. G Schematic illustration of wild-type (Wt) and mutant (Mut) sequences of one putative binding sites of TCF4 on ALOXE3 promoter are shown. H Transcriptional activity of ALOXE3 in Cal27 TCF4-knockdown or overexpression cells calculated by the luciferase reporter system. I Western blot to identify ALOXE3 and TCF4 protein level in Cal27 TCF4-knockdown or overexpression cells. ***P < 0.001, **P < 0.01, *P < 0.05.

Article Snippet: Following are the antibodies utilized for immunofluorescence and their concentrations: TRIB3 (Proteintech, China, 13300-1-AP, 1:100), β-catenin (Cell Signaling Technology, 2677 S, 1:200), TCF4 (NOVUS, H00006925-M03, 10 μg/ml), Anti-rat IgG (abcam, ab15007, 1:1,000), Anti-mouse IgG (abcam, ab150115, 1:1,000).

Techniques: Inhibition, Activity Assay, Knockdown, Control, ChIP-sequencing, Binding Assay, Over Expression, Mutagenesis, Luciferase, Western Blot

Journal: Cell death & disease

Article Title: TRIB3 promotes malignancy of head and neck squamous cell carcinoma via inhibiting ferroptosis.

doi: 10.1038/s41419-024-06472-5

Figure Lengend Snippet: Fig. 7 TRIB3‒β-catenin‒TCF4 heterotrimer complex negatively modulates ferroptosis by targeting ALOXE3. (A) Western blot to identify ALOXE3 protein level in TRIB3-knockdown or overexpression and control cells. (B) Western blot to measure ALOXE3 protein level in ALOXE3- knockdown and control cells. (C, D) Lipid peroxidation in ALOXE3-knockdown and control cells. (E) MDA in ALOXE3-knockdown and control cells. (F, G) Lipid peroxidation in HNSCC cells was transfected with TRIB3 and ALOXE3 shRNA. (H) MDA in HNSCC cells was transfected with TRIB3 and ALOXE3 shRNA. ***P < 0.001, **P < 0.01, *P < 0.05.

Article Snippet: Following are the antibodies utilized for immunofluorescence and their concentrations: TRIB3 (Proteintech, China, 13300-1-AP, 1:100), β-catenin (Cell Signaling Technology, 2677 S, 1:200), TCF4 (NOVUS, H00006925-M03, 10 μg/ml), Anti-rat IgG (abcam, ab15007, 1:1,000), Anti-mouse IgG (abcam, ab150115, 1:1,000).

Techniques: Western Blot, Knockdown, Over Expression, Control, Transfection, shRNA

Journal: Cell death & disease

Article Title: TRIB3 promotes malignancy of head and neck squamous cell carcinoma via inhibiting ferroptosis.

doi: 10.1038/s41419-024-06472-5

Figure Lengend Snippet: Fig. 8 Hesperidin suppresses HNSCC initiation and progression. A The active pocket of TRIB3 protein. B The structure of hesperidin. C Cell viability of HNSCC cells treated with different Hesperidin concentrations. D Western blot to detect TRIB3 protein level in HNSCC cells treated with hesperidin (20 μm) for 48 h. E MDA in HNSCC cells treated with hesperidin (20 μm) for 48 h. F, G Lipid peroxidation in HNSCC cells treated with hesperidin (20 μm) for 48 h. H The level of Fe2+ in HNSCC cells treated with hesperidin (20 μm) for 48 h. I Schematic of FaDu subcutaneous tumors treated with hesperidin, J, K tumor growth and L weight in the xenograft model. M The expression of Ki67 using IHC assay. N H&E staining of the spleen, heart, kidney, lung, and liver tissues. Scale bar: 100 μm. O The schematic diagram illustrates the inhibition of ferroptosis in HNSCC by the TRIB3-β-catenin-TCF4 complex through the suppression of ALOXE3 transcription. ***P < 0.001, **P < 0.01, *P < 0.05.

Article Snippet: Following are the antibodies utilized for immunofluorescence and their concentrations: TRIB3 (Proteintech, China, 13300-1-AP, 1:100), β-catenin (Cell Signaling Technology, 2677 S, 1:200), TCF4 (NOVUS, H00006925-M03, 10 μg/ml), Anti-rat IgG (abcam, ab15007, 1:1,000), Anti-mouse IgG (abcam, ab150115, 1:1,000).

Techniques: Western Blot, Expressing, Staining, Inhibition

Journal: Scientific Reports

Article Title: B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

doi: 10.1038/s41598-024-67912-y

Figure Lengend Snippet: Activated Cγ1 CD63-emGFP B cells undergoing Cre-mediated recombination in vitro express emGFP. ( a ) Schematic for Cγ1 CD63-emGFP reporter strain design. Transgenic mice expressing Cre recombinase driven by transcription of the Ig γ1 constant region gene segment (Cγ1) was crossed with a silenced reporter mouse, resulting in CD63-emerald GFP expression driven by the CAG promoter. ( b ) Percentage of emGFP + Cγ1 CD63-emGFP and Cγ1 Cre control B cells following stimulation with IL-4 or LPS + IL-4 for 3 days. Gates indicate GFP low/- B cells, GFP + B cells and GFP + B220 low/− B cells. ( c ) Percentage of cell surface expression of CD69, PNA and CD138 in GFP low/− , GFP + and GFP + B220 low/− B cells at day 3, for the Cγ1 CD63-emGFP reporter mice in panel ( b ). Gates were set on negative control samples (dotted black histograms). ( d ) Frequencies of IgG1 + B cells in B cells gated on GFP expression at days 3 and 7, for the reporter mice in ( b ). ( e ) Concentration of IgG1 in the culture medium of B cells from the reporter mice in ( b ). All data are expressed as mean ± SEM. Results shown are representative of at least three independent experiments. P = **0.01 and ***0.001, with unpaired, two-tailed t -test.

Article Snippet: Cγ1 CD63-emGFP B6 mice were subcutaneously immunized with influenza A virus PR8/34 recombinant HA protein (10 μg/mouse, Sino Biological, Wayne, PA) in the adjuvant Addavax on days 0, 14, and 21.

Techniques: In Vitro, Transgenic Assay, Expressing, Control, Negative Control, Concentration Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

doi: 10.1038/s41598-024-67912-y

Figure Lengend Snippet: In vivo generation of Cγ1 CD63-emGFP GC B cells and progeny IgG1 + memory B cells and plasma cells express emGFP after immunization with NP-KLH. ( a ) Flow cytometry gating strategy of GC B cells, IgG1 + SWM B cells, and plasma cells that express emGFP in the spleens of Cγ1 Cre and Cγ1 CD63-emGFP mice immunized with NP-KLH for 14 days. ( b ) Percentages of emGFP + cells within each gated B cell subset are shown. ( c ) Numbers of emGFP + B cells in spleens for the reporter mice shown in panel ( b ). ( d ) Immunofluorescence confocal microscopy of emGFP + B cells in the lymph nodes of mice immunized with NP-KLH for 14 days. ( e ) Serum total and NP-specific IgG1 antibody levels in naïve Cγ1 Cre and Cγ1 CD63-emGFP mice and after immunization with NP-KLH for 14 days. Data are expressed as mean ± SEM. Results shown in panels ( a – c ) and ( e ) are representative of three independent experiments and in panel d from > 20 images of two independent experiments. P = *0.05, **0.01, ***0.001 and ****0.0001, with unpaired, two-tailed t -test.

Article Snippet: Cγ1 CD63-emGFP B6 mice were subcutaneously immunized with influenza A virus PR8/34 recombinant HA protein (10 μg/mouse, Sino Biological, Wayne, PA) in the adjuvant Addavax on days 0, 14, and 21.

Techniques: In Vivo, Flow Cytometry, Immunofluorescence, Confocal Microscopy, Two Tailed Test

Journal: Scientific Reports

Article Title: B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

doi: 10.1038/s41598-024-67912-y

Figure Lengend Snippet: Activated Cγ1 CD63-emGFP B cells undergoing Cre-mediated recombination in vitro produce EVs that express emGFP, tetraspanins, and IgG. ( a ) Nanoparticle tracking analysis of total and GFP + EVs isolated from cell culture medium from Cγ1 CD63-emGFP B cells stimulated with LPS + IL-4 for 7 days. ( b ) EVs were visualized by cryoelectron microscopy. Images are representative of three independent EV preparations. ( c , d ) Lysates from EVs isolated from cell culture medium from Cγ1 CD63-emGFP B cells stimulated with IL-4, LPS, and LPS + IL-4 for 7 days were probed by Western blot for the presence of CD9, CD81, CD63, GFP, and IgG under non-reducing conditions, and IgG under reducing conditions (10 μg/lane). Under reducing conditions, IgG bands from EV samples 1 and 2 are juxtaposed from the same blot as indicated by vertical line, from full length blots shown in Supplementary Fig. b. Results show two EV preparations and are representative of at least six independent experiments. ( e ) ImageStream analysis of individual emGFP + EVs isolated from the cell culture medium of Cγ1 CD63-emGFP B cells stimulated with LPS + IL-4 and stained for surface IgG or CD16/32 (Alexa Fluor 647), CD64 (PE), and binding of exogenous mouse IgG (DyLight 405). ( f ) ImageStream analysis of peritoneal B cells stained for surface B220 (FITC) and binding of exogenous mouse IgG. ( g ) ImageStream analysis of peritoneal macrophages stained for surface CD11b (FITC) and binding of exogenous mouse IgG in the absence (left panel) or presence of 2.4G2 Fc block (right panel). The results shown are representative of three independent experiments.

Article Snippet: Cγ1 CD63-emGFP B6 mice were subcutaneously immunized with influenza A virus PR8/34 recombinant HA protein (10 μg/mouse, Sino Biological, Wayne, PA) in the adjuvant Addavax on days 0, 14, and 21.

Techniques: In Vitro, Isolation, Cell Culture, Cryo-Electron Microscopy, Western Blot, Staining, Binding Assay, Blocking Assay

Journal: Scientific Reports

Article Title: B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

doi: 10.1038/s41598-024-67912-y

Figure Lengend Snippet: Circulating EVs produced from Cγ1 CD63-emGFP B cells bind specific antigen and express GFP and EV tetraspanin markers after immunization with NP-KLH. ( a ) ELISA measurements of NP-specific total (NP32-BSA) and high-affinity (NP4-BSA) IgG antibody levels in whole serum (left two panels) and EVs isolated from serum (right two panels) from Cγ1 CD63-emGFP mice unimmunized or immunized with NP-KLH for 14 days. Sample dilutions shown are 1:1000–1:30,000 for serum and 3.3 × 10 7 –3.7 × 10 6 particles/ml for EVs. ( b ) ELISA measurements for the presence of both CD63 and CD9 and GFP from NP-specific EVs, for the reporter mice in panel ( a ). Data are expressed as mean ± SEM. The results shown are representative of three independent experiments.

Article Snippet: Cγ1 CD63-emGFP B6 mice were subcutaneously immunized with influenza A virus PR8/34 recombinant HA protein (10 μg/mouse, Sino Biological, Wayne, PA) in the adjuvant Addavax on days 0, 14, and 21.

Techniques: Produced, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Scientific Reports

Article Title: B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

doi: 10.1038/s41598-024-67912-y

Figure Lengend Snippet: HA-specific EVs neutralize influenza infection. ( a ) Experimental strategy for testing HA-specific EVs in influenza infection in vivo. ( b ) Four groups of mice ( n = 6 mice per group) were intranasally infected with PR8 virus only (Vehicle), PR8 virus mixed with high dose of EVs isolated from serum from Cγ1 CD63-emGFP mice immunized with influenza A hemagglutinin (HA) protein for 21 days (HA-high), PR8 virus mixed with low dose of EVs from the same mice immunized with HA (HA-low), or PR8 virus mixed with high dose of EVs isolated from serum from Cγ1 CD63-emGFP mice immunized with NP-KLH for 21 days. Host survival and body weight change following PR8 challenge were monitored. Data are expressed as mean ± SEM. The results shown are representative of two independent experiments. P = **0.001 between the HA-high and HA-low body weight change, calculated by unpaired, two-tailed t -test. P = * < 0.05 for survival in HA-high and HA-low groups as compared with NP-high and Vehicle groups, calculated by log-rank test. ( c ) Analysis of EV samples from serum from Cγ1 CD63-emGFP mice immunized with HA protein by size exclusion chromatography collected in 0.5 ml fractions. The number of particles per fraction was measured by nanoparticle tracking analysis and the amount of soluble IgG per fraction was measured by ELISA. Results shown are representative of two independent experiments.

Article Snippet: Cγ1 CD63-emGFP B6 mice were subcutaneously immunized with influenza A virus PR8/34 recombinant HA protein (10 μg/mouse, Sino Biological, Wayne, PA) in the adjuvant Addavax on days 0, 14, and 21.

Techniques: Infection, In Vivo, Virus, Isolation, Two Tailed Test, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

doi: 10.1038/s41598-024-67912-y

Figure Lengend Snippet: EVs derived from spontaneous germinal center B cell responses in Cγ1 CD63-emGFP Nba2 lupus-prone mice express surface IgG with self-reactivity. ( a ) Schematic for generating the Cγ1 CD63-emGFP reporter strain on the Nba2 lupus-prone congenic background. ( b ) Flow cytometry gating strategy of GC B cells, IgG1 + SWM B cells, and plasma cells that express emGFP in the spleens of 7-mo-old Cγ1 Cre Nba2 and Cγ1 CD63-emGFP Nba2 mice. ( c ) Percentages of emGFP + cells within each gated B cell subset are shown ( n = 9 mice per group). ( d ) Numbers of emGFP + B cells in spleens for the reporter mice shown in panel ( c ). ( e ) Immunofluorescence confocal microscopy of emGFP + B cells in the spleens of 7-mo-old Cγ1 CD63-emGFP Nba2 mice. ( f ) ELISA measurements of antinuclear IgG antibody levels in whole serum and EVs isolated from serum from Cγ1 CD63-emGFP Nba2 mice and Cγ1 CD63-emGFP B6 controls, for the reporter mice in panel ( c ). Sample dilutions shown are 1:500–1:3000 for serum and 6.6 × 10 8 –7.4 × 10 7 particles/ml for EVs. ( g , h ) ELISA measurements for the presence of antinuclear binding EVs that express either CD9 and CD81, CD63 and CD9, or CD63 and CD81, and emGFP from EVs, for the reporter mice in panel ( c ). Data are expressed as mean ± SEM. The results shown in panels ( b – d ) and ( f – h ) are representative of three independent experiments, and in panel e from two independent experiments. P = ***0.001 and ****0.0001, with unpaired, two-tailed t -test.

Article Snippet: Cγ1 CD63-emGFP B6 mice were subcutaneously immunized with influenza A virus PR8/34 recombinant HA protein (10 μg/mouse, Sino Biological, Wayne, PA) in the adjuvant Addavax on days 0, 14, and 21.

Techniques: Derivative Assay, Flow Cytometry, Immunofluorescence, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Isolation, Binding Assay, Two Tailed Test