EMBO/soda-nel huggingface dataset:
FLOW CYTOMETRY MULTIPLEXED METHOD FOR THE DETECTION OF NEUTRALIZING HUMAN ANTIBODIES TO THE NATIVE SARS-CoV-2 SPIKE PROTEIN (2021)
Figure 7

Figure 7. Prevalence of seropositivity by the FC Jurkat-S method within a cohort of 415 samples.

(A) Discrepancies between the result of the flow cytometry test and previous PCR or ELISA tests within the CBMSO cohort.

(B) A neutralization assay with S protein-pseudotyped lentivirus was carried out to resolve the discrepancies. Data represent the mean±SD of duplicates. *, p<0.05; **, p<0.005; ***, p<0.0005; *****, p<0.000005 (Paired two-tailed t-test comparing all serum dilutions to the pre-COVID sample). (C) Classification of the CBMSO cohort according to the Score generated from S/EGFR data. The Broken line indicates the 0.024 threshold above which samples are considered positive.

EXPANDED VIEW FIGURES
The immunity-related GTPase Irgm1 promotes the expansion of activated CD4+ T cell populations by preventing interferon-γ-induced cell death (2008)
figf1

(a) [3H]thymidine incorporation at 72 h by wild-type (WT) and Irgm1−/− CD4+ T cells activated with soluble mAb to CD3 and irradiated syngenic wild-type splenocytes. (b) [3H]thymidine incorporation at 72 h by OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with OVA protein (OVA) or OVA peptide of amino acids 323–339 (OVA(323–339)) in the presence of syngenic splenic DCs. (c,d) Kinetic analysis of [3H]thymidine incorporation (c) and cell numbers (d) in cultures of OT-II and Irgm1−/− OT-II spleen cells stimulated with OVA peptide. (e) Flow cytometric analysis of the activation and proliferation of OVA peptide–stimulated OT-II and Irgm1−/− OT-II CD4+ T cells pulsed with BrdU during the final 4 h of 72 h of culture and then stained with mAb to CD25 and BrdU. Numbers in quadrants indicate percent viable CD4+ cells. (f) Enzyme-linked immunosorbent assay of IL-2 produced by OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with OVA peptide in the presence of splenic DCs, measured at 72 h. Data are representative of more than five (a,b) or three (c–f) independent experiments with similar results (mean and s.d. of triplicate cultures, a–d,f).
The immunity-related GTPase Irgm1 promotes the expansion of activated CD4+ T cell populations by preventing interferon-γ-induced cell death (2008)
figf2

(a) Immunoblot analysis of Irgm1 in lysates of flow cytometry–sorted naive wild-type CD4+ lymphocytes activated for 48 h with immobilized agonistic mAb to CD3 (10 μg/ml) alone or with mAb to CD28 (2 μg/ml) in the presence or absence of IFN-γ-neutralizing mAb. Actin, loading control. (b) Enzyme-linked immunosorbent assay of IFN-γ produced by OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with OVA peptide in the presence of splenic DC in polarized conditions (TH1 or TH2) or nonpolarized conditions (N), assessed in 72-hour cultures. ND, not detected. (c) Accumulation of polarized and nonpolarized OT-II (open symbols) or Irgm1−/− OT-II (filled symbols) CD4+ T cells after peptide stimulation, quantified by counting of viable cells. (d) Viable OT-II and Irgm1−/− OT-II CD4+ T cells 72 h after restimulation with OVA peptide in the presence of T cell–depleted APC samples. (e) Flow cytometry of the viability and division of OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with peptide, allowed to 'rest' for 7 d in IL-2, then labeled with CFSE and restimulated with OVA peptide in the presence of T cell–depleted APC samples and assessed 72 h later. (f) Flow cytometric analysis of the population expansion of CFSE-labeled OT-II and Irgm1−/− OT-II CD4+ T cells activated with OVA peptide in the presence or absence of IFN-γ-neutralizing mAb. Results in e,f obtained after gating on CD4+ T cells; numbers in outlined areas (e) and in top left corners (f) indicate percent CD4+ cells. PI, propidium iodide. (g) [3H]thymidine incorporation by wild-type, Irgm1−/−, Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells stimulated for 72 h with soluble agonistic mAb to CD3 and irradiated syngenic Ifng−/− splenocytes. Data are representative of two independent experiments with similar results (mean and s.d. of triplicate cultures, b–d,g).
The immunity-related GTPase Irgm1 promotes the expansion of activated CD4+ T cell populations by preventing interferon-γ-induced cell death (2008)
figf3

(a) Flow cytometric analysis of the survival of CD45.2+ Ifng−/− (open symbols) or Irgm1−/−Ifng−/− (filled symbols) CD4+ T lymphocytes preactivated with agonistic mAb to CD3 in vitro, then transferred intraperitoneally into congenic CD45.1+ C57BL/6.SJL recipients left untreated or inoculated intraperitoneally with T. gondii or M. avium 3 d earlier, assessed in peritoneal exudate cell samples collected at day 4 after transfer. (b) Survival of M. avium–infected wild-type, Irgm1−/−, Ifng−/− and Irgm1−/−Ifng−/− mice (n = 5 mice per genotype). (c–e) Splenic bacterial loads (c), circulating lymphocytes (d) and splenic lymphocytes (e), assessed at week 4 after M. avium infection (n = 5 mice per genotype). Each symbol in a,c–e represents an individual mouse. Data are representative of two independent experiments.
The immunity-related GTPase Irgm1 promotes the expansion of activated CD4+ T cell populations by preventing interferon-γ-induced cell death (2008)
figf4

(a) [3H]thymidine incorporation by naive CD4+ lymphocytes stimulated for 72 h with agonistic mAb to CD3 and IFN-γ-deficient irradiated APCs, with IL-2 (10 U/ml) added 24 h after the initiation of culture. (b) Accumulation of viable CD4+ T cells at 72 h in the presence of medium alone (Med) or IFN-γ (5 U/ml). (c) Flow cytometric analysis of the viability and division of CD4+ T cells labeled with CFSE and activated as described in a with T cell–depleted Ifng−/− APC samples in the presence or absence of IFN-γ (5 U/ml). Numbers in plots indicate percent CFSE+CD4+ T cell populations. (d–f) Death of Ifng−/− and Irgm1−/−Ifng−/− T cell populations activated with mAb to CD3 and expanded in IL-2, then treated with IFN-γ (d), agonistic mAb to CD3 (e) or Fas ligand (FasL; f), assessed by propidium iodide staining at 48 h after treatment. Open bars, Ifng−/−; filled bars, Irgm1−/−Ifng−/− (a,b,d–f). Data are representative of three independent experiments with similar results (mean and s.d. of triplicate cultures a,b,d–f).
The immunity-related GTPase Irgm1 promotes the expansion of activated CD4+ T cell populations by preventing interferon-γ-induced cell death (2008)
figf5

(a–f) Electron microscopy analysis of Ifng−/− CD4+ lymphocytes (a) and Irgm1−/−Ifng−/− CD4+ lymphocytes (b–f) exposed for 30 h to IFN-γ (5 U/ml). (c) Enlargement of the area outlined in b; arrowheads indicate two autophagic vacuoles. (d) Quantification of vacuoles in 50 randomly selected electron microscopy sections of more than 400 cells. Each symbol represents one section. (e) Irgm1−/− CD4+ T cell with many vacuoles containing dense materials. (f) Two Irgm1−/− CD4+ cells with vacuolated cytoplasma. Original magnification (a–c,e,f), × 1,000. Data are representative of two experiments. (g,h) Death of Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells in the presence or absence of wortmannin or Ly294002 (g) and of cells transfected with control or beclin1-specific small interfering RNA, assessed by flow cytometry with propidium iodide staining at 48 h after IFN-γ exposure. Right (h), immunoblot analysis of beclin1 expression. C, control small interfering RNA; B, beclin1-specific small interfering RNA. Data are representative of three independent experiments (mean and s.d. of triplicate cultures).
Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry (2009)
figf1

(a) Fluorescence microscopy of GFP-LC3–transduced HeLa cells left unstimulated (Unstim) or stimulated for 4 h with C12-iEDAP (2.5 μg/ml) or for 2 h with rapamycin (Rapa; 50 μg/ml). Green, GFP-LC3; blue, DAPI (DNA-intercalating dye). Scale bar, 5 μm. (b) Quantification of dot- or ring-shaped GFP-LC3 signals (representing autophagosomes) in HeLa cells stimulated as described in a. (c) Confocal microscopy of wild-type and Nod2-deficient BMDMs transduced with GFP-LC3 (green) and left unstimulated or stimulated for 4 h with LPS (1 μg/ml) or MDP (100 μg/ml) or for 2 h with rapamycin (50 μg/ml). Scale bar, 15 μm. (d) Quantification of dot- or ring-shaped GFP-LC3 (green) in BMDMs stimulated as described in c. (e) Immunofluorescence microscopy of macrophages recovered from the peritoneal cavities of thioglycolate-injected wild-type, Nod1- and Nod2-deficient mice 4 h after intraperitoneal injection of MDP (300 μg/ml), FK565 (300 μg/ml) or rapamycin (300 μg/ml); after centrifugation by cytospin, cells were stained for endogenous LC3 (red) and with DAPI (blue). Scale bar, 10 μm. (f) Quantification of dot- or ring-shaped LC3 signals in macrophages recovered from the peritoneal cavities of mice treated as described in c. Data are representative of four (a–d) or two (e,f) independent experiments with at least 100 cells per condition (error bars (b,d,f), s.d.).
Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry (2009)
figf2

(a) Confocal microscopy of wild-type and Nod1-deficient GFP-LC3–expressing MEFs infected for 2 h with wild-type S. flexneri M90T-RFP. (b) Quantification of the colocalization of GFP-LC3 signals with bacteria in a. (c) Confocal microscopy of wild-type and Nod1-deficient GFP-LC3–expressing MEFs infected for 2 h with S. flexneri ΔIcsB-RFP. (d) Quantification of the colocalization of GFP-LC3 signals with bacteria in c. (e,f) Plate assay of bacteria in wild-type or Nod1-deficient MEFs infected for 0.5 h with S. flexneri ΔIcsB in the presence or absence of the autophagy blocker LY294002 (LY) or protease inhibitors (PI), followed by 5.5 h of incubation in the presence of gentamicin. (g–j) Confocal microscopy (g,i) and quantification of the colocalization of GFP-LC3 signals with M90T and ΔIcsB (h,j) in wild-type and RIP2-deficient (Ripk2−/−) GFP-LC3–expressing MEFs infected for 2 h with wild-type S. flexneri (M90T; g,h) or ΔIcsB S. flexneri (i,j). (k) Enzyme-linked immunosorbent assay of KC secretion by wild-type and RIP2-deficient MEFs infected for 0.5 h with M90T and incubated for 5.5 h in the presence of gentamicin. (l,m) Confocal microscopy (l) and quantification of the colocalization of GFP-LC3 signals (green) with M90T and ΔIcsB (m) of wild-type and NEMO-deficient (Ikbkg−/−) GFP-LC3–expressing MEFs infected for 0.5 h with wild-type S. flexneri (M90T) or S. flexneri ΔIcsB and incubated for 1.5 h in the presence of gentamicin. Red, RFP. Arrows indicate colocalization of GFP-LC3 signals with S. flexneri. Scale bars, 10 μm. *P 0.05, and **P 0.01 (t-test). Data represent three independent experiments with at least 100 cells each (error bars, s.e.m.).
Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry (2009)
figf3

(a) Immunofluorescence of HeLa cells expressing hemagglutinin-tagged Nod1 (HA-Nod1 (red); top) or HA-Nod2 (red; bottom). Blue, DAPI. (b) Immunofluorescence of HeLa cells expressing Myc-tagged FL-ATG16L1 (Myc–FL-ATG16L1). (c,d) Immunofluorescence of HeLa cells coexpressing HA-Nod1 (red; c) or HA-Nod2 (red; d) and Myc–FL-ATG16L1 (green). Blue, DAPI. (e) Immunofluorescence of HeLa cells expressing a Flag-tagged truncated form of ATG16L1 (Flag–ΔN85-ATG16L1; green). Blue, DAPI. (f) Immunofluorescence of HeLa cells coexpressing HA-Nod2 (red) and Flag–ΔN85-ATG16L1 (green). Blue, DAPI. (g) Immunofluorescence microscopy of Mode-K cells stained for endogenous Nod2 (green) and ATG16L1 (red) and with DAPI (blue). For a–g, arrows indicate membrane colocalization of ATG16 and Nod2; area outlined at left is enlarged at right (a,b,e) or in other images (c,d,f,); scale bars, 8 μm. (h,i) Immunoprecipitation (IP) and immunoblot (IB) analysis of HEK293 cells transfected with Flag–ΔN85-ATG16L1 (h) or Myc–FL-ATG16L1 (i), together with HA-Nod1, HA-Nod2 or HA-tagged enhanced GFP (EGFP-HA). Ig, immunoglobulin (loading control). Results are representative of one of at least three independent experiments (a–g) or one of three independent experiments (h,i).
Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry (2009)
figf4

Immunofluorescence microcopy of wild-type and RIP2-deficient MEFs expressing Flag–tagged ΔN85-ATG16L1 and HA-Nod1 (a) or HA-Nod2 (b). Scale bars, 10 μm. Area outlined at far left is enlarged at right. Data are representative one of two independent experiments.
Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry (2009)
figf5

(a) Fluorescence microscopy of immortalized GFP-LC3–transduced lymphoblasts from donors homozygous for the normal ATG16L1*300T allele (300T) or risk allele ATG16L1*300A (300A), left unstimulated or treated for 2 h with rapamycin (50 μg/ml) or for 4 h with MDP (20 μg/ml). Green, GFP-LC3. Scale bar, 5 μm. (b) Induction of GFP-LC3 signals by rapamycin, MDP or Gram-positive peptidoglycan (PG+; 20 μg/ml) in cells treated as described in a, presented relative to GFP-LC3 signals in unstimulated control cells. *P 0.05 (t-test). Data represent three independent experiments (error bars (b), s.d.).
Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry (2009)
figf6

(a) Immunofluorescence microscopy of HeLa cells transfected with HA-Nod2 (red) and Flag–ΔN85-ATG16L1(green) and infected for 10–20 min with S. flexneri M90T. Blue, DAPI. (b) Immunofluorescence microscopy of endogenous Nod2 and ATG16L1 in Mode-K cells infected as described in a. DIC, differential interference contrast; blue, DAPI. Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria. Scale bars, 5 μm. Data are representative of one of three independent experiments.
Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry (2009)
figf7

(a,b) Immunofluorescence microscopy of HeLa cells expressing Flag–ΔN85-ATG16L1 (green; a) or Myc–FL-ATG16L1 (green; b) and HA-tagged Nod2 L1007fsinsC (HA–Nod2, 1007fs). Area outlined at left is enlarged in other images. (c,d) Immunofluorescence microscopy of HeLa cells expressing Flag–ΔN85-ATG16L1 (c) or Myc–FL-ATG16L1 (d) together with HA-tagged Nod2fs (red). Blue, DAPI. Scale bar, 8 μm. (e) Immunofluorescence microscopy of wild-type and Nod2fs-KI BMDMs transduced with GFP-LC3 (green) and infected for 0.5 h with S. flexneri M90T-RFP (red), followed by incubation for 1 h with gentamicin. Arrows indicate no colocalization of Nod2 with ΔN85-ATG16L1 or FL-ATG16L1. Scale bar, 15 μm. (f) Quantification of the colocalization of GFP-LC3 signals with S. flexneri M90T-RFP in experiments as described in e. Data are representative of one of three independent experiments (error bars (f), s.d.).
TRPA1 and TRPV1 contribute to iodine antiseptics-associated pain and allergy (2016)
Figure 1

Figure 1. TRPA1 and TRPV1 mediate iodine-induced pain in mice.

A. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine. In this and subsequent similar figures, the number of mice is indicated.

B. Averaged intracellular Ca2+ signals in cultured mouse DRG neurons in response to consecutive applications of 0.25 ppm iodine, 30 μM AITC and 2 μM capsaicin. All of the iodine responsive neurons (n = 332) from 6 adult mice were included in the analysis.

C, D. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine, following the intraperitoneal injection of HC030031 (HC) (C), AMG517 (D) or vehicle.

E. Quantification of the nociceptive responses within 5 min after intraplantar injection of control saline in WT mice or 500 ppm iodine in WT and TRPA1-/- mice.

F. Quantification of the nociceptive responses in TRPA1-/- mice within 5 min after intraplantar injection of control saline or 1,000 ppm iodine, following intraperitoneal injection of AMG517 or vehicle.

Data are presented as mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001 (Two-tailed Student's t-test).
TRPA1 and TRPV1 contribute to iodine antiseptics-associated pain and allergy (2016)
Figure 2

Figure 2. Iodine in PVP-I promotes contact hypersensitivity in mice mainly through TRPA1 and TRPV1.

A. Left, time course of ear swelling elicited by topical application of 5% PVP-I solution in 0.15% Oxa-challenged WT and TRPA1-/- mice, or 5% povidone solution in Oxa-challenged WT mice. In this and subsequent similar figures, the number of mice is indicated. Right, bar graph highlighting the response at 24 h shown at left.

B. Time course of ear swelling elicited by topical application of the indicated concentrations of iodine in 0.15% Oxa-challenged mice, and a bar graph highlighting the response at 24 h.

C. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged WT and TRPA1-/- mice, and bar graph highlighting the response at 24 h.

D. Hematoxylin and eosin stained tissue sections of iodine treated ear in 0.15% Oxa-challenged WT and TRPA1-/- mice (n ≥ 3).

E. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged TRPA1-/- mice, following intraperitoneal injection of AMG517 or vehicle, and bar graph highlighting the response at 24 h.

F. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged mice, following intraperitoneal injection of RP67580 or vehicle, and bar graph highlighting the response at 24 h.

Data are presented as mean ± s.e.m. Statistical significance was evaluated using two-tailed Student's t-test (for all two-group comparisons) or one-way analysis of variance (ANOVA) followed by Tukey test (for multi-group comparisons).

*P<0.05, **P<0.01, **P<0.001.
TRPA1 and TRPV1 contribute to iodine antiseptics-associated pain and allergy (2016)
Figure 3

Figure 3. Iodine activates TRPA1 but not TRPV1.

A. Representative intracellular Ca2+ signals in hTRPA1-expressing HEK 293 cells in response to different concentrations of iodine. 2-APB, a TRPA1 agonist, was subsequently applied to fully activate TRPA1. RFU: relative fluorescence unit.

B. Concentration-response relationships of iodine-induced intracellular Ca2+ increase in HEK 293 cells expressing WT or mutant TRPA1 channels. Data are presented as mean ± s.e.m. n ≥ 8 for each construct at each concentration. The smooth curves are fits to the Hill equation.

C. Time course of iodine-induced currents in hTRPA1-expressing Xenopus oocytes. HC: HC030031

D. Concentration-response relationship of iodine-induced currents in hTRPA1-expressing Xenopus oocytes. Data are presented as mean ± s.e.m. n ≥ 8 for each concentration. The smooth curve is a fit to the Hill equation.

E. Time course of intracellular iodine-induced macroscopic currents in an inside-out patch from hTRPA1-expressing HEK 293 cell (n = 3).

F. Time course of iodine-induced currents in a hTRPA1-expressing Xenopus oocyte, which presents the current reduction upon DTT treatment (n = 3).

G, H. Comparisons of the iodine-induced whole-cell current in HEK 293 cells expressing WT hTRPA1 (G) or the triple mutant channel (C421S/K710R/C856S) (H) (n ≥ 4).

I. Representative whole-cell currents in HEK 293 cells expressing human TRPV1 in response to iodine and subsequently applied capsaicin (n = 3).
MacroH2A1.2 deficiency leads to neural stem cell differentiation defects and autism-like behaviors (2021)
Figure 1

Figure 1 mH2A1.2 is expressed in the developing brain

(A) mH2A1.2 and different neurogenesis markers are present in cortical lysates collected at various time points (E13, E15, E17, and P0).

(B) Immunofluorescence staining for mH2A1.2 in the cerebral cortex at E13, E15 and P0. Scale bar represents 100μm.

(C) A scatter diagram shows the change trend of mH2A1.2 and different neurogenesis markers. n = 5 mice, independent replicates.

(D) mH2A1.2 is expressed in neural progenitor cells and neurons cultured in vitro. Scale bar represents 50 μm.

(E) Electroporation of mH2A1.2-shRNA results in abnormal cell distribution in the developing neocortex. Electroporation was performed at E13.5, and the mouse was harvested at E16.5. Scale bar represents 50 μm.

(F) Graph shows the percentage of GFP-positive cells distributed in the VZ/SVZ, IZ and CP. n = 7 mice, independent replicates.

Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.01(**) or P < 0.001(***). n.s., not significant.
MacroH2A1.2 deficiency leads to neural stem cell differentiation defects and autism-like behaviors (2021)
Figure 2

Figure 2 Loss of mH2A1.2 increases proliferation of neural progenitor cell.

(A) Schematic of the mH2A1 knockout mouse construction strategy. The H2Afy1 gene encoded mH2A1 was knocked out by the CRISPR-Cas9 method.

(B) Western blot analysis of mH2A1 expression levels in WT and mH2A1 KO cortices at E13.5.

(C) Graph shows the normalized density of mH2A1.2 versus control. n = 6 mice, independent replicates.

(D)Abnormal cell distribution was observed in the mH2A1.2 silenced neocortex. The GFP plasmid was electroporated into WT and KO mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm.

(E) Graphs of the percentage of GFP-positive cells in the VZ/SVZ, IZ and CP.

n = 5 mice, independent replicates.

(F) Brain sections of WT and KO mice at E16.5 were immunostained with the mitotic marker pH3 and DAPI. Scale bar represents 20 μm.

(G) Statistics of pH3+ cells of the cortex. n=6 mice, independent replicates.

(H) Representative images of E16.5 coronal brain sections were immunostained for PAX6. The GFP plasmid was electroporated into WT and KO mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm.

(I) Quantification of GFP and Pax6 double positive cells in the VZ/SVZ. n=6 mice, independent replicates.

Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.