EMBO/soda-nel huggingface dataset:
Potent virus neutralization by human IgM against SARS-CoV-2 is impaired by class switch (2022)
Figure 2

Figure 2. Spike-capturing B cells from peripheral blood of convalescent donors.

A-D. Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies shown in Appendix Table S4 and measured by flow cytometry. (A) UMAP algorithm (5000 randomly selected cells/sample) was used to depict the major B cell subsets clustered according to marker expression. FlowSOM-based B cell subpopulations are overlaid as a color dimension, and the colors of the clusters are shown on the left of heatmap (B). Cells falling in the MACAC gate (see below) are marked with red rhombi.

B. Heatmap showing mean population expression levels of markers used for UMAP visualization and FlowSOM-clustering. Colors shown in the legend on the left are also used in the UMAP representation in (A).

C. Gating strategy to define CD69-high, mCherry-high, spike-capturing B cells (i.e., membrane antigen capture activated cells, MACAC, red box). Dot plot shows CD69 and mCherry fluorescence for B cells from one of three donors gated by forward and side scatter, and negative for the dye used to mark the antigen donor cells.

D. Relative fractions of different B cell subpopulations, from three donors, as shown in A-B, in all B cells compared to within MACAC B cells. P values are based on two-tailed t-tests between the groups. Correlation coefficients (r) were calculated from the z-statistic of the Wilcoxon-Mann-Whitney test. A black horizontal line represents the median. Boxplots represent the interquartile range (IQR). Whiskers extend to the farthest data point within a maximum of 1.5× IQR. Every point represents one donor.

E. Gating strategy in MACAC sorting. Single cells are selected based on scatter, antigen-expressing TE spike-mCherry cells excluded on Cell Trace Violet label, and the spike-capturing (mCherry-high), activated (CD69-high) B cells (population labeled "MACAC" in red on the right-most plot) are sorted. The middle plot shows B cells that did not adhere to the TE-spike-cherry antigen-expressing cells (putatively antigen-irrelevant), and the right plot those that did (putatively antigen-recognizing). Plots show data from one of five convalescent donors (those whose serum results are labeled with black triangles in Fig 1A). Cells in the MACAC gate were singly distributed into wells of 384-well plates and cultured for 9 days with IL-21 and CD40L, and then the single well culture supernatants were screened for anti-spike antibody binding and virus neutralization as described below.

F. Results of single well supernatant screening for antibody binding to SARS-CoV-2 spike protein. Results from 3266 wells from 5 donors are shown for IgM (left), IgG (middle), and IgA (right). Screening and plotting methods are the same as in (Fig 1A).
A gene regulatory network controls the balance between mesendoderm and ectoderm at pluripotency exit (2019)
Figure 1

Figure 1. Comparison of gene expression changes during mESC differentiation towards the three germ layers.
A. Immunostaining of TUJ1, DESMIN and GATA6 after six days of mESC differentiation towards ectoderm, mesoderm and endoderm. Bar: 50 μm.
B. Scheme of the experimental approach to quantitatively capture gene expression changes during mESC differentiation towards the three germ layers at high temporal resolution.
C. Venn diagram of genes with differential expression during mESC differentiation towards the three germ layers.
D-F. Hierarchical clustering of mRNA expression of mESCs differentiated towards ectoderm (E), mesoderm (F) and endoderm (G).
A gene regulatory network controls the balance between mesendoderm and ectoderm at pluripotency exit (2019)
Figure 5

Figure 5. A Sox1-Brachyury-Eomes triple knock-in mESC line to simultaneously monitor germ layer specification
A. Triple knock-in (3KI) mESC line that simultaneously reports on ectoderm fate, (marked by the expression of Sox1::GFP), mesendoderm fate (marked by the expression of T::3xTagBFP, T is also known as Brachyury), or definitive endoderm fate (marked by the expression of Eomes::mCherry).
B. Ectoderm differentiation of the 3KI mESC line (marked by the expression of Sox1::GFP).
C. Mesendoderm differentiation of the 3KI mESC line (marked by the expression of T::3xTagBFP).
D. Endoderm differentiation of the 3KI mESC line (marked by the expression of Eomes::mCherry).
E, F. Spontaneous differentiation of the 3KI mESC line leading to the coexistence of cells belonging to the three germ layers. Bar: 100 μm.
A gene regulatory network controls the balance between mesendoderm and ectoderm at pluripotency exit (2019)
Figure 6

Figure 6. Determining the role of the highly connected nodes on mESC differentiation
A. Scheme to assess the influence of the highly connected nodes on germ layer acquisition.
B. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zfp354c-/- mESCs in the 3KI background to ectoderm.
C. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zfp354c-/- mESCs in the 3KI background to mesendoderm.
D. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zff354c-/- mESCs in the 3KI background to definitive endoderm.
E. Scheme to assess the influence of the highly connected nodes on the spontaneous fate acquisition between mesendoderm and ectoderm.
F. Differentiation of wild type (WT), Sp1+/-, Nr5a2-/- and Fos-/-Jun-/- mESCs in the 3KI background to definitive endoderm.
Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus. (2015)
Figure 1

The calcineurin inhibitor FK506 impairs innate immunity to Aspergillus fumigatus

A C57BL/6 WT or Rag2−/− mice were immunosuppressed with hydrocortisone +/− FK506 and infected with 105 AF CEA10 conidia intranasally. Mortality was assessed using a 20% weight loss endpoint. Mortality was significantly increased in FK506 immunosuppressed mice reaching 100% by day 10 p.i. (P = 0.003 for HC versus HC+FK506 in Rag2−/− using log-rank test, n = 10 per group).

B, C PAS-stained lung sections of Rag2−/− mice were scanned and inflammation quantified by pixel intensity threshold analysis in ImageJ. FK506-treated animals had increased inflammation at day 2 p.i. compared to control. P = 0.0126 (using Student's t-test), n = 3-5 per group. (C) Close-up of an infectious focus showing increased inflammation and fungal growth in the FK506-treated group. Scale bar: 400 μm.

D, E Murine macrophages (J7741.A) were pre-treated with hydrocortisone, and the effects of FK506 on nuclear shuttling of the glucocorticoid receptor were assessed by confocal immunofluorescence microscopy. The nuclei are indicated in red, and the GR is indicated in green. Scale bar: 50 μm. FK506 did not cause increased translocation of the glucocorticoid receptor. Data represent mean + SEM calculated from 10 fields of vision. One out of 2 experiments is shown.
Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus. (2015)
Figure 2

Calcineurin inhibition leads to impaired cytokine production, reduced early neutrophil recruitment and delayed fungal killing during pulmonary aspergillosis

Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n.

A Weight was monitored daily for 7 days p.i. Immunosuppressed mice showed increased weight loss through days 1–3 p.i. Data points represent mean ± SEM.

B BALs were performed over a 3-day period p.i. and fungal killing assessed by enumerating CFUs. Fungal killing was significantly impaired in immunosuppressed mice. Error bars indicate SEM.

C, D Flow cytometry analysis of the CD45+ leucocyte populations in BAL. Representative dot plots for CD45+ cells (C) and percentages of Ly-6G-positive neutrophils infiltrating the infected lungs over a 48-h p.i. time course (D). The bars indicate the mean percentage (± SEM) of neutrophils of total CD45+ leucocytes (n = 5 mice per group). Neutrophil influx was significantly impaired in immunosuppressed mice at 6 h p.i.

E PAS-stained lung sections of immunocompetent and FK506-treated animals at 6 h p.i. Scale bar: 50 μm. IC animals showed inflammatory cell infiltrates around conidia (black arrows), whereas in FK506-treated animals, only uptake of conidia by the resident macrophage population could be seen.

F Tissue density of whole lung sections was quantified by pixel intensity analysis using ImageJ. Bars represent mean percentages (± SEM), n = 3.

G Luminex multiplex cytokine analysis of BAL SNs at 6 h p.i. Bars represent mean ± SEM.

Data information: For statistical analysis, individual time points were compared using Student's t-test.
Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus. (2015)
Figure 3

Calcineurin inhibition impairs neutrophil recruitment in a zebrafish model of invasive aspergillosis

At 2 days post-fertilization (dpf), Lyz:dsRed or mpx:GFP larvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafish larvae were microinjected with AF eGFP conidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed.

A, B Absolute neutrophil numbers in treated and untreated mpx:GFP larvae were quantified by direct counting of fluorescent neutrophils in non-infected whole fish (n = 11-13 per group). There were no significant differences.

C For survival analysis, lyz:dsRed larvae were microinjected with ˜10 conidia and the effects of FK506 on the outcome from invasive aspergillosis was determined by Kaplan–Meier analysis for survival over 6 days post-infection (n = 16 per group). Immunosuppression led to a significant increase in mortality.

D TNF-α mRNA levels were measured in whole non-infected or infected larvae 48 h p.i. Immunosuppression led to significant and almost complete inhibition of TNF-α expression during infection.

E-G Lyz:dsRed larvae were infected with ˜50 eGFP-expressing conidia of A. fumigatus and were imaged daily from 24 h p.i. (E) The effects of FK506 on neutrophil recruitment were assessed by integrated density analysis of red fluorescent neutrophils at sites of infection in ImageJ. A representative image showing impaired neutrophil recruitment to the site of infection in immunosuppressed larvae is shown (60 h p.i.). The arrows indicate infectious foci. (F) Neutrophil recruitment was minimal throughout the first 60 h p.i. in immunosuppressed larvae (n = 8 per group). (G) Fungal growth was determined by measurement of the length of individual fluorescent hyphal elements (n = 8 per group).

Data information: All data represent mean ± SEM and were statistically analysed by Student's t-test.
Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus. (2015)
Figure 4

FK506 impairs calcineurin-dependent TNF-α responses to A. fumigatus in murine macrophages

A J774A.1 macrophages were treated with an increasing concentration of FK506 and stimulated with AF SC (MOI = 1), zymosan (50 μg/ml) or LPS (25 ng/ml) overnight. TNF-α was measured in the culture supernatant by ELISA. FK506 significantly reduced TNF-α levels at 5 and 10 ng/ml for AF. A significant reduction in TNF-α was also seen for zymosan, but not LPS.

B J774A.1 cells were treated with FK506 (10 ng/ml) and either left non-stimulated (NS) or stimulated with AF SC (MOI=1) for 6 h. TNF-α mRNA levels were quantified from whole-cell RNA extracts by RT–PCR. FK506 significantly reduced TNF-α expression.

C J774A.1 macrophages were treated with control (Ctrl.) or calcineurin A (CnA)-targeting siRNA (50 nM) and stimulated with either AF SC (MOI = 1), zymosan (50 μg/ml) or LPS (25 ng/ml) overnight. TNF-α was measured in the culture supernatant by ELISA. CnA siRNA led to significant reductions in both AF- and zymosan-dependent TNF-α levels, but had no significant effect on LPS responses.

Data information: Multiple comparisons for (A) were performed after a two-way ANOVA, and comparison for (B, C) was carried out by Student's t-test. ns, not significant; NS, non-stimulated. *P < 0.05; **P < 0.01. Data represent mean + SEM of three independent experiments.Source data are available online for this figure.
Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus. (2015)
Figure 5

Aspergillus fumigatus induces sustained NFAT responses in murine macrophages

A J774A.1 macrophages were stimulated with live AF SC (MOI=5), and macrophage cytoplasmic and nuclear extracts were probed by Western blot for NFATc2. NFATc2 shuttling to the nucleus was observed and was maximal at 2 h p.i.

B J774A.1 macrophages were stimulated with live or fixed swollen conidia (MOI = 5) or zymosan (50 μg/ml), and NFAT translocation was assessed by confocal microscopy. NFAT is indicated in green, the nuclei are indicated in red, and AF conidia can be seen in pink. Live conidia were observed to stimulate sustained nuclear translocation compared to dead conidia or zymosan. Data represent mean ± SEM. Scale bar: 50 μm. Arrows indicate A. fumigatus or zymosan-positive cells.

C J774A.1 macrophages were stimulated with zymosan after FK506 (10 ng/ml) or control treatment, and macrophage nuclear extracts were probed by Western blot for NFATc2 and NF-κB translocation. NFATc2 and NF-κB nuclear translocation was observed, which was maximal at 1 h p.i., in stimulated macrophages. FK506 specifically inhibited NFATc2 translocation, but had no effect on NF-κB.

D J774A.1 macrophages were pre-treated with FK506 (10 ng/ml) or vehicle and stimulated with live swollen conidia or zymosan. NFATc2 translocation was evaluated 30 min after stimulation. Box and whisker plot shows median ± maximum/minimum. Scale bar: 50 μm. FK506 completely inhibited nuclear translocation of NFATc2 in response to AF or zymosan. Statistical analysis was carried out by Student's t-test. NS, non-stimulated. Arrows indicate A. fumigatus or zymosan-positive cells.

Source data are available online for this figure.
Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus. (2015)
Figure 6

Phagocytosis is required for A. fumigatus-induced NFAT activation through a Dectin-1–Syk- and MyD88-independent pathway

A BMDMs from WT, Dectin-1−/− or MyD88−/− mice were infected with swollen conidia (MOI = 1), and TNF-α was measured in the SN after overnight incubation. TNF-α levels were significantly reduced in Dectin1−/− and Myd88−/− BMDMs.

B BMDMs were stimulated with swollen conidia (MOI = 5) or zymosan (50 μg/ml), and NFATc2 translocation was quantified by confocal microscopy 30 min after stimulation. Whilst almost complete abrogation of NFAT translocation was seen in Dectin1−/− macrophages stimulated with zymosan, NFAT responses to AF were unaffected.

C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (C) TNF-α was measured in the SN after overnight incubation. Syk inhibition significantly reduced swollen conidia-dependent or zymosan-dependent TNF-α levels. (D) NFATc2 translocation was quantified by confocal microscopy. Syk inhibition impaired zymosan-dependent NFAT translocation, but did not affect AF-dependent NFAT translocation. (E) Whole-cell lysates of Syk or Ctrl. siRNA-treated cells were separated by SDS–PAGE, followed by Western blotting, and membranes were probed with anti-Syk and anti-β-actin antibodies.

F J774A.1 macrophages were pre-treated with piceatannol and infected with swollen conidia (MOI = 5). Phagocytosis was quantified by FACS 30 min after stimulation. Syk inhibition led to impaired phagocytosis of zymosan, but had no effect on phagocytosis of SC.

G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (G) NFATc2 translocation after 30 min of stimulation was quantified by confocal microscopy. (H) For quantification of NF-κB translocation, nuclear extracts were separated by SDS–PAGE, followed by Western blotting. Membranes were probed with anti-NF-κB p65 and anti-HDAC1 antibodies. (I) After overnight stimulation, TNF-α was measured in the SN by ELISA. Inhibition of phagocytosis with cytochalasin D led to impaired NFAT translocation in response to zymosan or AF, but not ionomycin (G). In addition, inhibition of phagocytosis lead to increased translocation of NF-κB (H) and increased levels of TNF-α in response to either zymosan or AF.

Data information: Bar graphs represent mean ± SEM and box and whisker plots represent median ± maximum/minimum value. Statistical analysis was performed using (A, B, C) two-way ANOVA for multiple comparisons and (D–I) Student's t-test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. NS, non-stimulated; UT, untreated. Source data are available online for this figure.
Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus. (2015)
Figure 7

TLR9 mediates MyD88-independent endosomal NFAT activation

A J774A.1 macrophages were pre-treated with bafilomycin A1 (100 nM) and stimulated with swollen conidia (MOI = 3) for 30 min. NFATc2 translocation was assessed by confocal microscopy. Acidification was required for NFATc2 translocation.

B TLR9 recruitment was assessed in WT BMDMs 30 min after infection with eGFP-expressing swollen conidia by confocal microscopy. Scale bar: 10 μm. The arrow indicates a TLR9-positive AF-containing phagosome.

C BMDMs from WT or TLR9−/− mice were infected with swollen conidia (MOI = 1), and after overnight stimulation, TNF-α was measured in the SN by ELISA. TLR9−/− BMDMs had significantly lower TNF-α levels.

D, E BMDMs from WT or TLR9−/− mice were infected with swollen conidia (MOI = 5) for 30 min, and NFATc2 translocation was quantified by (D) confocal microscopy and (E) Western blotting of nuclear extracts. TLR9−/− BMDMs exhibited reduced NFATc2 translocation by both approaches.

F J774A.1 macrophages were pre-treated with the TLR9-blocking nucleotide ODN2088 (10 μM) for 30 min, and NFATc2 translocation was quantified by confocal microscopy. This led to a significant reduction in NFATc2 translocation in response to AF.

G BMDMs from WT or MyD88−/− mice were pre-treated with ODN2088 (10 μM) and infected with swollen conidia (MOI = 5) for 30 min. NFATc2 translocation was quantified by confocal microscopy. In both, WT and MyD88−/− BMDMs, blocking TLR9 significantly impaired NFATc2 translocation.

Data information: Bar graphs represent mean + SEM and box and whisker plots represent median maximum/minimum value. All statistical analyses were performed using Student's t-test. NS, non-stimulated. One representative out of three experiments is shown. Source data are available online for this figure.
Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus. (2015)
Figure 8

Bruton's tyrosine kinase mediates TLR9-dependent calcineurin activation

A, B J774A.1 macrophages were pre-treated with the BTK inhibitor LFM-A13 (12.5 μM) and infected with swollen conidia. (A) After overnight stimulation, TNF-α was measured in the SN by ELISA. (B) After 30 min of stimulation, NFATc2 translocation was quantified by confocal microscopy. Both TNF-α levels and NFATc2 translocation were significantly reduced by BTK blockade.

C J774A.1 macrophages were treated with a control or BTK-targeting siRNA (25 nM), and NFAT translocation was quantified by confocal microscopy. BTK knock-down led to a significant reduction in NFATc2 translocation in response to AF.

D, E J774A.1 macrophages were treated with a control, BTK (25 nM)- or TLR9 (75 nM)-targeting siRNA and additionally pre-treated with either ODN2088 (10 μM) or the BTK inhibitor LFM-A13 (12.5 μM). The cells were infected with swollen conidia (MOI = 5), and NFATc2 translocation was measured by confocal microscopy. Blocking both TLR9 and BTK signalling had no additive effect on NFAT translocation blockade.

Data information: Bar graphs represent mean ± SEM and box and whisker plots represent median ± maximum/minimum value. All statistical analyses were performed using Student's t-test. NS, non-stimulated. Source data are available online for this figure.
Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus. (2015)
Figure 9

TNF-α responses to A. fumigatus in human alveolar macrophages are dependent on both NF-κB and NFAT signalling

Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage. Cells were pre-treated for 1 h with FK506 (10 ng/ml), the NFAT inhibitor MCV1 (1 μM) or SC514 (10 μM) before stimulation with A. fumigatus swollen conidia (MOI 0.1). After 24 h of stimulation, TNF-α was measured in the cell culture supernatant by ELISA.

A Representative FACS-phenotyping demonstrative of a normal alveolar macrophage phenotype is shown for three patient samples.

B Inhibition of alveolar macrophage responses to AF with FK506, MCV1 or SC514 led to a significant reduction in TNF-α levels.

Data information: Data represent mean ± SEM. Statistical test used is Kruskal–Wallis with Dunn's multiple comparison. *P < 0.05, ***P < 0.001. Each data point represents an individual donor.
Chronic cold exposure enhances glucose oxidation in brown adipose tissue (2020)
Figure 2

Fig. 2, Chronic cold exposure induces oxidative metabolism in sWAT.

(A) Representative immunofluorescence staining shows UCP1 (green), perilipin (red) and DAPI (blue) in sWAT of mice housed at 6°C.

(B-G) Male mice, housed at 30°C or 6°C for 10 days, were administered with [U-13C]glucose (2 g/kg, IP). 15 minutes after injection, sWAT and gWAT were harvested for metabolic enrichment assay. Metabolic 13C enrichments in sWAT of male mice are shown as m+6 glucose (B), m+3 glycolytic intermediates (C), and m+2 TCA cycle intermediates (D). Metabolic 13C enrichments in gWAT of male mice are shown as m+6 glucose (E), m+3 glycolysis intermediates (F), and m+2 TCA cycle intermediates (G).

(H) Plasma 13CO2 enrichment, after normalizing to the [U-13C]glucose tracer enrichment.

Data information: n=10 mice per group for A-G, n=7-8 mice per group for H, data are represented as the mean ± s.d. Statistical analysis was performed using two-tailed Student's t-test, *P < 0.05.
Chronic cold exposure enhances glucose oxidation in brown adipose tissue (2020)
Figure 4

Fig. 4, β3-AR agonist activates glucose oxidation in differentiated primary brown adipocytes.

(A) Fully differentiated brown adipocytes were treated with β3-AR agonist CL316,243 (10 µM). Relative mRNA levels of thermogenic markers were measured by qPCR.

(B,C) (B) Pyruvate and lactate enrichment, (C) Citrate and malate enrichment were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose and 10 µM CL316,243 for 2 hours.

(D) Results of metabolic flux analysis (MFA) of primary brown adipocytes treated with or without CL316,243. Arrows are weighted according to absolute flux magnitudes. See Appendix Table S1 for definitions of quantitative flux values, and the units of the flux values are nmol/h/μg protein. OAA, oxaloacetate.

Data information: n=3 biological repeats, data are represented as the mean ± s.d. Statistical analysis was performed using two-tailed Student's t-test (A), two-way ANOVA followed by Tukey's multiple comparisons test (B and C), *P < 0.05.