EMBO/soda-nel huggingface dataset:
Cyprinid-specific duplicated membrane TLR5 senses dsRNA as functional homodimeric receptors (2022)
Figure 7

Figure 7. N-glycosylation is necessary for CiTLR5a and CiTLR5b-mediated IFN1 and NF-κB2 signal transduction, but not ligand binding.

Ribbon diagrams of the structure models of CiTLR5a and CiTLR5b ectodomains. The predicted N-glycosylation sites were labeled. The conserved Asn (N) residues between CiTLR5a and CiTLR5b are shown in pink, the specific Asn residues in CiTLR5a and CiTLR5b are shown in blue and orange, respectively.

Comparison of predicted N-glycosylation sites between CiTLR5a and CiTLR5b. The conserved Asn residues were marked by triangles. The differentiated residues were denoted with gray background. "-" represents non-N-glycosylation site in the corresponding sequence.

Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b were respectively treated with Endo H and analyzed by immunoblotting (IB) with anti-HA antibody.

Effects of tunicamycin (5 μg/mL) on CiTLR5a- and CiTLR5b-mediated NF-κB2 (D) and IFN1 (E) signalings were investigated by DLR assays in FHM cells (n = 3). HsTLR5 and CiMDA5 were employed as positive controls, respectively.

Mutants with the predicted N-glycosylation sites were transfected into FHM cells and examined via immunoblotting to show the change of molecular weight.

FHM cells were respectively transfected with mutants and the indicated receptor combinations, and then stimulated with poly(I:C) or FlaC. Vector and wild type (WT) were used as controls. IFN1 (G) and NF-κB2 (H) promoter activities were examined by DLR assays (n = 3).

I-L Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b mutants were respectively incubated with biotin-poly(I:C) (I) or FlaC (K). Vector and wild type were used as controls. Pull-down and immunoblotting assays were conducted (I, K). The images of (I) and (K) were one of the three independent biological experiments in Appendix Fig S4A and B. (J, L) Relative band intensity ratios of (I) and (K) were analyzed by ImageJ (n = 3).

Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (D), (E), (G), (H), (J), and (L). n represents biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.
Cyprinid-specific duplicated membrane TLR5 senses dsRNA as functional homodimeric receptors (2022)
Figure 8

Figure 8. M/M type TLR5 sense poly(I:C) but not other types.

Lysates of FHM cells overexpressing LoTLR5 (A), DrTLR5a (B), DrTLR5b (B), IpTLR5-1 (C), IpTLR5-2 (C), IpTLR5S (C), OmTLR5M (D), OmTLR5S (D), and HsTLR5 (E) were respectively incubated with FlaC-His (1 μg/mL) at pH 5.0 or pH 7.4. Complexes were pulled down by Anti-GFP antibody coupling beads and analyzed by immunoblotting (IB) with anti-His antibody.

Lysates of FHM cells overexpressing LoTLR5 (F), DrTLR5a (G), DrTLR5b (G), IpTLR5-1 (H), IpTLR5-2 (H), IpTLR5S (H), OmTLR5M (I), OmTLR5S (I), and HsTLR5 (J) were respectively incubated with biotin-poly(I:C) (1 μg/mL) at pH 5.0 or pH 7.4. Complexes were pulled down by streptavidin beads and analyzed via immunoblotting with anti-GFP antibody.

Effects of DrTLR5a and DrTLR5b constructs on IFN1 (K), IFN3 (L), and NF-κB2 (M) promoter activities were measured by DLR assays in FHM cells (n = 3).

Lysates of FHM cells overexpressing DrTLR5a and DrTLR5b mutants were incubated with biotin-poly(I:C) and analyzed by immunoblotting (N), and relative band intensity ratios were analyzed by ImageJ (O) (n = 3). Vector and wild type (WT) were used as controls. The images of (N) were one of the three independent biological experiments in Appendix Fig S6A and B.

Summarized models of TLR5-ligand interaction and immune responses. Upper panel: The solid lines represent that ligand can be recognized by the corresponding types of TLR5, and the dotted lines represent that ligand cannot bind to the corresponding types of TLR5. Lower panel: The arrows indicate that different combinations of duplicated membrane TLR5 promote signal transduction after stimulation by the corresponding ligand, the dotted lines indicate that different combinations bind the corresponding ligand (flagellin) but do not transmit signals. The "⊥" symbol indicates that different combinations slightly suppress signal transduction after stimulation by poly(I:C).

Representative TLR5 protein sequences were employed to construct the phylogenetic tree by neighbor-joining method within the MEGA11 program. The tree was bootstrapped 10,000 times.

Summarized model of TLR5 evolution in vertebrates.

Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (K-M) and (O). n represents biological replicates. *P < 0.05, **P < 0.01.

Expanded View Figure Legends
A Norrin/Wnt surrogate antibody stimulates endothelial cell barrier function and rescues retinopathy (2021)
Figure 1

Figure 1. F4L5.13 treatment activates the Wnt-ßcatenin pathway in LEF/TCF reporter cell lines and cultured endothelial cells.

A. Top: Molecular architecture of tetravalent F4L5.13. Bottom: Schematic for activation of Wnt/ßcatenin signaling by F4L5.13.

B. Activation of ßcatenin signaling by F4L5.13 or recombinant NDP (30nM each) in HEK293T cells transfected with FZD4 and/or LRP5. Values represent fold-activation of LEF/TCF reporter gene. Data are presented as mean ± SEM, n=3.

C. Dose response curves for the activation of a LEF/TCF reporter gene in HEK293T cells transfected with FZD4, LRP5 and with either GFP or TSPAN12 by serial dilutions of F4L5.13 or NDP proteins (x-axis). Data are presented as mean ± SEM, n=3.

D. RT-qPCR of Axin2 expression in bEnd.3 cells treated with serial dilutions of F4L5.13, NDP or isotype control for 24h. Data are presented as mean ± SEM, n=3.

E. RT-qPCR of Axin2 in bEnd.3 cells treated with NDP (200 ng/ml), isotype control or F4L5.13 (1200 ng/ml) and transfected with control, Tspan12 or Fzd4 siRNAs. Data are presented as mean ± SD, n=3 technical replicates. Data is representative of two independent experiments.

F. Time course of phosphorylated Disheveled-3 (p-DVL3) and ßcatenin protein levels in bEnd.3 cells treated with 30nM of F4L5.13 or NDP. Histogram represents the ratio of the DVL3 phosphorylation levels over total DVL3 protein and ßcatenin levels over ß-Tubulin measured by densitometry of independent experiments. Data are presented as mean ± SEM, n=4-6 (*p<0.05 as compared with NT). Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared with NT).
A Norrin/Wnt surrogate antibody stimulates endothelial cell barrier function and rescues retinopathy (2021)
Figure 3

Figure 3. F4L5.13 promotes endothelial cell barrier function.

A. Expression of endothelial junction protein ZO-1 and stabilization of ßcatenin in bEnd.3 cells treated with 30 nM F4L5.13 or 30 nM recombinant NDP for the indicated times. Blots are representative of five different experiments. Histogram represents the ratio of ZO-1 and ßcatenin levels over ß-Tubulin measured by densitometry of five independent experiments. Data are presented as mean ± SEM. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared with NT).

B. Immunofluorescence of ZO-1, CLDN3 and CLDN5 localization at bEnd.3 cell junctions. bEnd.3 cells were treated or not with 30 nM F4L5.13 or 30 nM Norrin (NDP) with or without 100 ng/ml VEGF for 1h. ZO-1 is shown in green, CLDN3/5 in red and DAPI in blue. Scale bars: 15 µm.

C. Quantification of ZO-1, CLDN3 and CLDN5 fluorescence intensity. Each column represents 40 measurements of fluorescence intensity per condition (y-axis). Data are presented as mean ± SEM. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared to VEGF treatment).

D. Transendothelial permeability assay quantifying the passage of FITC-dextran through a monolayer of bEnd.3 cells. Passage of FITC-dextran was measured after exposure of bEnd.3 cells to 100 ng/ml VEGF, 30 nM F4L5.13 or both or pretreated with VEGF for 1h before treating with F4L5.13 for 1h. Data are presented as mean ± SD, n=5 independent experiments. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared to VEGF treatment).
A Norrin/Wnt surrogate antibody stimulates endothelial cell barrier function and rescues retinopathy (2021)
Figure 4

Figure 4. F4L5.13 restores intraretinal angiogenesis and blood-retina barrier formation in Tspan12-/- mice.

A. F4L5.13 prevents BRB defects and restores vascular morphogenesis in Tspan12-/- mice. 10 mg/kg F4L5.13 or isotype control antibody was injected i.p. every 48h from P5 to P20. Multiple images acquired at 10x magnification were stitched together to create a high-resolution image of a representative part of the retinal flat mounts. Scale bar 500 µm.

B. Optical sections of IB4-stained P20 retinas showing the mouse three-layered retinal vasculature (superficial, intermediate, and deep layers at 20x magnification) to document blood vessel formation or malformation. In Tspan12-/- mice, the intermediate capillary layer fails to properly develop and glomeruloid vascular malformations are present. The deep capillary layer is virtually absent. F4L5.13 fully restores intraretinal capillary development in Tspan12-/- mice. Scale bars: 100 µm.

C. IB4-stained retinal cross sections show the restored retinal vasculature and the absence of misdirected angiogenesis after F4L5.13 treatment. Boxed areas are shown enlarged in the panels on the right. Scale bars: 500 µm.

D. Quantification of the vascularized area in the outer plexiform layer. 10 mg/kg F4L5.13 or isotype control antibody was injected every 48h from P5 to P20. Data are presented as mean± SD, n=5 retinas (2-4 fields of view per retina) from 4-5 mice per group. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test.

E. F4L5.13 rescues barrier function defects in Tspan12-/- mice as shown by an increased expression of the tight junction component CLDN5 and decreased expression of the EC fenestration component PLVAP. IB4-Alexa647 was used to stain ECs. Scale bars: 100 µm.
A Norrin/Wnt surrogate antibody stimulates endothelial cell barrier function and rescues retinopathy (2021)
Figure 5

Figure 5. F4L5.13 partially restored BRB function in mature Tspan12-/- mice without any effect on the aberrant vascular morphology.

A. Injection schema for blood-retina barrier assay in adult Tspan12-/- mice with established retinal vascular defects.

B. Quantification of extravasated FITC-dextran in retinal lysates from WT and Tspan12-/- mice injected with isotype control antibody or F4L5.13. Data are presented as mean± SEM, n=6 retinas from 3 mice per group. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared to Tspan12-/- control treatment).

C. Quantification of the vascularized area in adult Tspan12-/- mice injected with isotype control or F4L5.13. Data are presented as mean± SD, n=4 retinas (4 fields of view per retina) from 2 mice per group. p-value was calculated by Student's t‐test.

D. Flat-mounted retinas of adult Tspan12-/- mice injected systemically with sulfo-NHS-biotin show that F4L5.13 partially restored BRB function. PECAM was used to stain endothelial cells. Images are representative of 4 retinas per group. Scale bar: 500 µm.

E. Maximum intensity projection of adult Tspan12-/- retinas after mice were injected with F4L5.13 or isotype control. Endothelial cells were stained with IB4-Alexa 647. Images are representative of 4 retinas per group. Scale bar: 100 µm.
A Norrin/Wnt surrogate antibody stimulates endothelial cell barrier function and rescues retinopathy (2021)
Figure 6

Figure 6. A FZD4:LRP5 antibody agonist reduces pathological neovascularization in the OIR model.

A. Schematic diagram of the OIR model (top) and representative images of P17 OIR retinas are shown (bottom). Neonatal mice were exposed to 75% oxygen from P7 to P12 to induce vessel loss. Then mice received intravitreal injections of PBS vehicle, mouse-specific anti-VEGF (as a positive control) delivered at 0.1 μg/μl, or F4L5.13v2 (a second-generation F4L5.13 modality, in which the N-terminal FZD4-specific diabody was replaced with a FZD4-specific Fab) at 50nM or 500nM target vitreous concentrations. Mice were returned to room air from P12 to P17 to induce maximum pathologic neovascularization at P17. At P17, retinas from injected mice were collected, dissected as flat‐mounts and stained with Isolectin B4. Scale bar = 500 µm.

B. Quantification of the ratio of neovascular and avascular area on total area of vascularization. Data are presented as mean ± SEM, n=11-13 retinas per group. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (indicated p-values are in comparison to Vehicle).

Expanded View Figure legends
FLOW CYTOMETRY MULTIPLEXED METHOD FOR THE DETECTION OF NEUTRALIZING HUMAN ANTIBODIES TO THE NATIVE SARS-CoV-2 SPIKE PROTEIN (2021)
Figure 1

Figure 1. Jurkat-S cells expressing native SARS-CoV-2 spike S protein allow the detection of anti-S protein antibodies in human sera.

(A) Lentiviral construct used to permanently express S protein in Jurkat. The full length mature S protein is preceded by a leader sequence of GM-CSF and followed by a T2A sequence which is followed by a tail-less truncated human EGFR construct.

(B) Expression of S protein on the plasma membrane of Jurkat-S cells assessed by surface biotinylation and followed by immunoprecipitation with anti-T2A, SDS-PAGE under reducing conditions and western blot with streptavidin-peroxidase. Biotinylation of the parental Jurkat cells was carried out in parallel as a negative control. The arrow indicates the position of the reduced S protein.

(C) Expression of the S protein in native form was assessed by surface biotinylation of Jurkat-S and Jurkat cells followed by immunoprecipitation with anti-T2A followed by SDS-PAGE under non-denaturing conditions (i.e. without reducing agents and without boiling). The nitrocellulose membrane was blotted with streptavidin-peroxidase. Arrows point at different oligomerization forms of the S protein.

(D) Formation of syncytia between the CTFR-labelled ACE2+ human cell line and the CFSE-labelled Jurkat-S cells was measured by flow cytometry by analyzing the percentage of cells that become double positive for CTFR and CFSE markers. The Bar plot to the right shows the effect of different doses of HepG2 cells on the formation of syncytia with a fixed number of Jurkat. Parental Jurkat cells (not expressing S protein) are considered negative controls. Data represent the mean±SD of triplicated datasets.

(E) Formation of syncytia between the CTFR-labelled ACE2+ human cell line and the CFSE-labelled Jurkat-S cells, in an experiment as in D, was confirmed by confocal microscopy after sorting cells double positive for CTFR and CFSE. Nuclei were identified by DAPI staining. Red arrowhead in the DAPI image indicates a nucleus of Hep-G2 origin; the green arrowhead the nucleus of Jurkat-S origin.

(F) Overlay plot of Jurkat-S cells that were incubated with anti-EGFR mAb conjugated to Bv421 and serum from either donor #15 or from a pre-COVID-19 donor and followed by a secondary anti-human IgG1 antibody conjugated to PE.
FLOW CYTOMETRY MULTIPLEXED METHOD FOR THE DETECTION OF NEUTRALIZING HUMAN ANTIBODIES TO THE NATIVE SARS-CoV-2 SPIKE PROTEIN (2021)
Figure 2

Figure 2. Flow cytometry of Jurkat-S cells allows to detect anti-S immunoglobulins poorly detected by commercial tests with a wide dynamic range.

(A) Overlay histograms of Jurkat-S staining with different human sera diluted 1:50. A pre-COVID19 serum sample is taken as negative control (grey histogram).

(B) Bar plots of flow cytometry data generated with Jurkat-S cells and the panel of serum samples of Table EV1 classified according to their result in the indicated commercial tests: green, positive for the test; orange, weak or unclear; magenta, negative samples. Flow cytometry data is expressed as the ratio between the MFI of the antibody anti-S and the MFI for EGFR. Negative values for the flow cytometry test are those with a S/EGFR MFI ratio lower than 0.5. This ratio was set in order to fit most of the data negative for the other serological tests (pink triangles) under that threshold.Data represent the mean±s.e.m. All datapoints are shown.
FLOW CYTOMETRY MULTIPLEXED METHOD FOR THE DETECTION OF NEUTRALIZING HUMAN ANTIBODIES TO THE NATIVE SARS-CoV-2 SPIKE PROTEIN (2021)
Figure 3

Figure 3. Flow cytometry of Jurkat-S cells detects anti-S antibodies in sera otherwise negative for ELISA using recombinant S1 and RBD proteins.

(A) Box and whiskers plot of MFI and absorbance data in human sera according to their clinical classification of COVID-19 symptoms. Clinical classification was done according to the following parameters: Asymptomatic, no symptoms; Mild, 3 or more of the following symptoms: non-productive cough, hyperthermia, headache, odynophagia, dyspnea, asthenia , myalgia, ageusia, anosmia, cutaneous involvement; Moderate, 3 or more of the above symptoms plus gastrointestinal symptoms, or more than 3 of the above for 7 or more days; Moderate-Severe, 3 or more of the above symptoms plus pneumonia. Box and whiskers are shown to represent the minimum and maximum values as well as the median. All datapoints are shown.

(B) Comparison of MFI versus absorbance data generated by flow cytometry and ELISA for all human sera. A lineal regression curve was adjusted, with a 95% confidence interval, to all data with the R2 values indicated in the plots. #5Selected samples of outliers (#4, #5, #8, #31, #46, #48, #49 and #58) as well as samples close to a diagonal (#36, #52 and #15) are indicated.

(C) Titration of selected human sera by ELISA using the S1 protein and by flow cytometry with Jurkat-S cells. Samples with antibodies detected by flow cytometry and not by ELISA are indicated (#8, #46, #48, #49). Data represent the mean±SD of duplicates. An unpaired two-tailed t-test was carried out to compare the results of 1:50, 1:150 and 1:450 dilutions of sera #8, #46, #48 and #49 by FC to the pre-COVID-1 sample as a negative control. *, p<0.05; **, p<0.005; ***, p<0.0005.
FLOW CYTOMETRY MULTIPLEXED METHOD FOR THE DETECTION OF NEUTRALIZING HUMAN ANTIBODIES TO THE NATIVE SARS-CoV-2 SPIKE PROTEIN (2021)
Figure 4

Figure 4. Sera not detected by ELISA but detected by flow cytometry with Jurkat-S contain neutralizing antibodies.

(A) Cartoon of the strategy for generation of pseudotyped lentiviruses. HEK-293T cells were transfected with a lentiviral construct containing the EGFP marker gene and two more plasmids encoding for the gag and pol genes of HIV-1 and for the S protein of SARS-CoV-2. Alternatively, the latter plasmid was replaced by another one encoding the G protein of VSV. The cell supernatants collected after 48 h of transfection were used to transduce HEK-293T cells stably transfected with human ACE2.

(B) Validation of the neutralization method was carried out with cell HEK-293T cell culture supernatants containing lentiviral particles pseudotyped with either the S protein or with the VSV G protein. The supernatants were mixed with sera from donor #66 at the indicated dilutions or from a pre-COVID sample as a control before addition onto HEK-293T-ACE2+ cell cultures. No serum, refers to a control containing no human sera. Data represent the mean±SD of triplicates.

(C) The presence of neutralizing antibodies in sera from donors #46 and #48, found negative by ELISA and positive by flow cytometry (Fig. 3C) was demonstrated as in panel B. Data represent the mean±SD of triplicates. *, p<0.05; **, p<0.005; ****, p<0.00005 (Paired two-tailed t-test comparing all serum dilutions to the pre-COVID sample).

(D) Bar plot showing the S/EGFR MFI ratio determined by flow cytometry analysis with Jurkat-S of serum samples from 30 sanitary personnel repeatedly tested as PCR negative for SARS-CoV-2 at the Ramón y Cajal Hospital of Madrid. A negative result is considered for a ratio lower than 0.5. Two clear cases of sera positive for anti-S IgG1 are indicated (RyC52 and RyC65). A borderline sample just above the threshold line (RyC58) is also indicated. Data show the mean±s.e.m. All datapoints are shown.

(E) Two-color dot plot overlay of anti-S protein fluorescence versus EGFR fluorescence for borderline serum sample RyC58 (blue) and the clearly negative RyC70 sample (red). The line plot to the right shows the MFI for EGFR taken at the 3 sectors indicated in the two-color plot and the corresponding MFI values for EGFR.

(F) Plot of the S/EGFR MFI ratio for the 30 RyC samples versus a Score generated according to the slope of S/EGFR and shape (see Materials and Methods and Fig. EV1). The green line indicates the threshold point (0.024) for the Score above which a serum is considered positive.

(G) The presence of neutralizing antibodies in sera from donors RyC46, RyC52, RyC56 and RyC58 diluted 1/15 was demonstrated as in panel B. Data represent the mean±SD of triplicates. *, p<0.05; **, p<0.005; ****, p<0.00005 (Unpaired two-tailed t-test comparing each serum to the No serum sample)
FLOW CYTOMETRY MULTIPLEXED METHOD FOR THE DETECTION OF NEUTRALIZING HUMAN ANTIBODIES TO THE NATIVE SARS-CoV-2 SPIKE PROTEIN (2021)
Figure 5

Figure 5. Validation of the FC Jurkat-S method.

(A) A total of 52 serum samples from Hospital Universitario La Princesa (HUP) and 52 samples of pre-COVID donors stored at the CBMSO were tested by the flow cytometry method and classified according to the S/EGFR-based Score. Of the 52 HUP samples, a total of 40 were from donors testing positive by PCR and by an ELISA test, 6 were from donors testing negative by PCR but positive by ELISA, 1 from an individual testing negative by ELISA and positive by PCR, and 5 were not tested by PCR. Box and whiskers are shown to represent the minimum and maximum values as well as the median. All datapoints are shown. The broken line indicates the threshold for a Score value of 0.024.

(B & C) Four HUP serum samples giving discrepant results by PCR and by the flow cytometry method were tested in the neutralization assay of pseudotyped lentivirus (Fig. 4A). Data represent the mean±SD of duplicates. *, p<0.05; **, p<0.005; ****, p<0.00005; (Paired two-tailed t-test comparing all serum dilutions to the pre-COVID sample)
FLOW CYTOMETRY MULTIPLEXED METHOD FOR THE DETECTION OF NEUTRALIZING HUMAN ANTIBODIES TO THE NATIVE SARS-CoV-2 SPIKE PROTEIN (2021)
Figure 6

Figure 6. Evolution of the presence of anti-S antibodies between the months of June and October 2020.

(A) A collection of serum samples from volunteers working at the CBMSO were taken in the months of June and October 2020 and tested by flow cytometry using Jurkat-S cells at the indicated dilutions. Flow cytometry data are shown as the S/EGFR MFI ratio. Red arrows indicate a loss in titer of antibodies between June and October whereas blue arrows indicate an increase in antibody titer.

(B) Titration of the neutralizing capacity of sera from a high titer (#66-CBM-1) and a low titer (CBM-2) donor taken in June and October 2020. The neutralization experiment was carried out in parallel to a FC Jurkat-S test. Flow cytometry results are shown as the S/EGFR MFI ratio (right y-axis) and according to the Score value. A plus symbol (+) indicates a Score >0.024 for a given serum dilution; a minus symbol (-) indicates a Score <0.024. Neutralization data are shown as the percenther of HEK-293T-ACE2+ cells becoming GFP+ (left y-axis). Data represent the mean±SD of duplicates. *, p<0.05; **, p<0.005; ***, p<0.0005; ****, p<0.00005; *****, p<0.000005 (unpaired two-tailed t-test).