EMBO/soda-nel huggingface dataset:
Human soluble ACE2 improves the effect of Remdesivir in SARS-CoV2 infection (2020)
Figure 1

Figure 1. Blocking entry and replication of SARS-CoV-2 infections

(A,B) (A) Remdesivir and (B) hrsACE2 inhibition of SARS-CoV-2 infections of Vero E6 cells. Both drugs, and murine recombinant soluble ACE2 (mrsACE2, control treatment), were used at the indicated concentrations. Viral RNA level was determined by qRT-PCR 15 hours after inoculation of SARS-CoV-2 (Swedish isolate, 106 PFU).

Data information: Error bars show mean ±SD from biological triplicate. n= 3, *: p<0.05; **: p<0.01; One-way ANOVA followed by student t-test between internal groups. P-values are listed in Appendix Table S1.

Source data are available online for this figure.
Human soluble ACE2 improves the effect of Remdesivir in SARS-CoV2 infection (2020)
Figure 3

Figure 3. Combined effect of remdesivir and hrsACE2 in blocking entry and replication of SARS-CoV-2 infections

(A) Treatment of SARS-CoV-2 (106 PFU) infected Vero-E6 cells with Human recombinant soluble ACE 2 (hrsACE2) (200µg/ml) and remdesivir (Remd. 4µM). Viral RNA level was determined at 15 hours after virus inoculation.

(B) Treatment of SARS-CoV-2 (106 PFU) infected human kidney organoids with hrsACE2 (200µg/ml) and/or remdesivir (Remd.4µM)). Viral RNA was determined by qRT-PCR 72 hours after the inoculation of 106 PFU of SARS-CoV-2.

(C) Treatment of SARS-CoV-2 (106 PFU) infected Vero-E6 cells with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM)).

(D) Treatment of SARS-CoV-2 (106 PFU) infected kidney organoids with hrsACE2 (10µg/ml) and remdesivir (4µM).

(E,F) Progeny virus released from untreated Vero-E6 cells or Vero-E6 cells treated with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM). Progeny was determined (E) 15 hours and (F) 48 hours post-infection (hpi).

Data information: Error bars show mean ±SD from biological triplicate. n= 3, *: p<0.05; **: p<0.01; ***:p<0.001; ****: p<0.0001; One-way ANOVA followed by student t-test between internal groups. P-values are listed in Appendix Table S1.

Source data are available online for this figure.
Murine Mpdz-Linked Hydrocephalus is Caused by Hyperpermeability of the Choroid Plexus (2018)
Figure 1

Fig. 1: Hydrocephalus was detected by PET in Mpdz-/- mice. A. Images of P4 (top) and P21 (bottom) Mpdz+/+ and Mpdz-/- mice. Arrowheads point to the domed foreheads of the latter. B. Mean weights of P18-P21 Mpdz+/+ and Mpdz-/- mice (n=8÷12, mean ± SD). C. Coronal, axial, and sagittal (top to bottom) PET images of P18-P21 Mpdz+/+ and Mpdz-/- mice. The emission intensity is shown as a 7-point temperature scale from black (0) to white (7). D. Mean PET emission intensities in the indicated brain sections (n=4, mean ± SD).
Murine Mpdz-Linked Hydrocephalus is Caused by Hyperpermeability of the Choroid Plexus (2018)
Figure 2

Fig. 2: Severe hydrocephalus and leakage of contrast medium were detected in Mpdz-/- mice by MRI. A. Coronal, sagittal, and axial (clockwise from top left corner image of each genotype) MR images of Mpdz+/+ and Mpdz-/- P18-P21 mice. Ventricles are pseudo-colored in red and 3D-reconstructed in the lower left panels. B. Means of total ventricle volumes and total brain volumes of Mpdz+/+ and Mpdz-/- mice (n=8, mean ± SD). C. A coronal HE-stained section (center) flanked by anatomically-corresponding coronal T2-weighted MR images of Mpdz-/- P18-P21 mice. Arrows or arrowheads show the match between the lateral ventricle CP in the HE section and in the MR images. D. Duplicate rows of T2-weighted coronal images and anatomically-corresponding T1-weighted coronal images at 1 min and at the peak-signal time point after contrast-medium injection; the bottom row shows a similar set of axial images. Areas surrounded by squares are magnified in the insets; arrows mark the contrast-medium signal in the T1-weighted images. Each row corresponds to one mouse aged 18-21-days. E. Time courses of the normalized T1-weighted image intensities corresponding to the areas surrounded by numbered squares on the T2-weighted images. Scale bars, 1 mm; insets, 0.5 mm.
Murine Mpdz-Linked Hydrocephalus is Caused by Hyperpermeability of the Choroid Plexus (2018)
Figure 3

Fig. 3: The Sylvian aqueduct of the Mpdz-/- mouse is stenotic. A. Coronal HE-stained brain sections of a P18 Mpdz+/+ mouse (out of a total of three) showing the 3rd ventricle, and the proximal and distal sections of the aqueduct of Sylvius; these features are marked by arrows in the insets. B. The same, for P18 Mpdz-/- mice. C. Top views of brains of P7 Mpdz+/+ and Mpdz-/- mice that show the sites of Evans blue injection, and the midline planes (dashed line) sectioned to produce the sagittal images below. They show the extent of Evans blue spread in the ventricles and surrounding tissue (one out of three experiments). In, injection; LV, lateral ventricle; SA, Sylvian Aqueduct; V3, third ventricle; V4, fourth ventricle. Scale bars, 1 mm; insets, 0.25 mm.
Murine Mpdz-Linked Hydrocephalus is Caused by Hyperpermeability of the Choroid Plexus (2018)
Figure 4

Fig. 4: Mpdz was localized proximal to the apical surface of CPECs. A. Immunofluorescence images of 10 μm-thick sections of CP from third ventricle villi of Mpdz+/+ and Mpdz-/- mice were immunolabeled as shown. The areas in the square frames are magnified in the bottom panels. B, C. CP sections from lateral ventricle villi immunolabeled for ZO1 (B) or E-cadherin (E-cad; C). The images are representative of 2 Mpdz+/+ mice and 3 Mpdz-/- mice. Scale bars, top panels, 100 μm; bottom panels, 10 μm. D. Immunoblots with the indicated antibodies (top) and their quantifications. The densitometry measurements were normalized relative to the signal of the samples transduced by non-targeting shRNA (Ctrl). Note that the same β-actin immunoblot was used twice because the Jam-C and E-cadherin samples were immunoblotted on the same membrane (mean ± SD, n=3).
Murine Mpdz-Linked Hydrocephalus is Caused by Hyperpermeability of the Choroid Plexus (2018)
Figure 5

Fig. 5: CPECs of Mpdz-/- mice harbored structural defects. A. TEM images of longitudinal sections of lateral ventricle CP villi of P18-P21 Mpdz+/+ and Mpdz-/- mice. Scale bar, 2 μm. B. A large number of voids of varying sizes was evident in the CPECs of Mpdz-/- mice. Higher magnification images of the areas in the rectangles are shown underneath the panels. Adherens junctions are denoted by horizontal lines and arrows; voids are indicated by arrows. Scale bar, 1 μm; insets, 200 nm. C. Triplicate images of tight junctions proximal to the apical faces of CPECs in Mpdz+/+ and Mpdz-/- mice. Scale bar, 100 nm D. Mitochondria were smaller and frequently lacked large portions of their cristae. Autophagosomes are present in some of them (arrows). Scale bar, 200 nm. The images are representative of 2 Mpdz+/+ mice and 2 Mpdz-/- mice. E. Time course and standard deviations of the impedance of confluent hpCPEC monolayers that were transduced by either MPDZ or non-targeting (Ctrl) shRNA. Each record represents 4 wells. MPDZ immunoblot of each cell group is shown in the inset.
Murine Mpdz-Linked Hydrocephalus is Caused by Hyperpermeability of the Choroid Plexus (2018)
Figure 6

Fig. 6: Fluid-phase uptake by CPECs is higher in Mpdz-/- relative to Mpdz+/+ mice. A. TEM images of CPEC sections from lateral ventricle villi of two Mpdz+/+ and two Mpdz-/- P14-P16 mice injected with HRP. The CPs were reacted with hydrogen peroxide and DAB ex-vivo. The dark particles are DAB deposits internalized by micropinocytosis. The magnified fields to the right show individual particles engulfed in macropinosomes. Note the layered structure of the particles, and the macropinosome that is open to the ventricular space in the Mpdz-/- section. Scale bars, 1 μm; insets, 100 nm. B. Mean number of engulfed DAB particles per cell in Mpdz+/+ and Mpdz-/- mice (mean ± SD, n=22). C. A CPEC section showing the engulfment of a DAB particle by its basal ruffles in the magnified field. Scale bars in panels C and D, 1 μm; insets, 200 nm. D. A CPEC section showing a preponderance of macropinosomes close to the apical face of the cell, and a magnified field that contains several macropinosomes. E. Mean numbers of DAB-containing macropinosomes close to the apical or basal sides of CPECs from Mpdz+/+ or Mpdz-/- mice (mean ± SD, n=20).
Murine Mpdz-Linked Hydrocephalus is Caused by Hyperpermeability of the Choroid Plexus (2018)
Figure 7

Fig. 7: LDLR abundance and endocytosis by CPECs are higher in Mpdz-/- relative to Mpdz+/+ mice. A. Immunofluorescence images of 10 μm-thick sections from lateral ventricle CP villi of Mpdz+/+ and Mpdz-/- P14-P16 mice immunolabeled by anti-LDLR. Scale bars, 50 (top) and 25 (bottom) μm. B. Mean fluorescence intensities (normalized relative to the highest recorded intensity) per cell in several LDLR-immunolabeled CP sections (mean ± SD, n=101). C. Immunoblots showing the abundances of LDLR and MPDZ in hCPECs transduced by MPDZ-targeting or non-targeting shRNA. Mean abundances are quantified by densitometry of the LDLR bands, normalized relative to the β-actin bands (mean ± SD, n=3). D. Fluorescence images of hCPECs transduced by either MPDZ or non-targeting (Ctrl) shRNA and immunolabeled by anti-LDLR either before (0 min) or after 8 min of constitutive endocytosis of LDLR. The histograms below show the mean fluorescence intensities per hCPEC in each cell group, normalized relative to the highest recorded intensity (mean ± SD, n=41÷53).
Murine Mpdz-Linked Hydrocephalus is Caused by Hyperpermeability of the Choroid Plexus (2018)
Figure 8

Fig. 8: The protein content in the CSF of Mpdz-/- mice was higher than in the CSF of Mpdz+/+ mice. A. Mean protein concentration in the CSF of Mpdz+/+ and Mpdz-/- P14-P21 mice (mean ± SD, n=2÷3). B. Volcano plot showing that the protein contents in sera of P15-P21 Mpdz+/+ and Mpdz-/- mice are similar to each other. C. A volcano plot showing that 23 proteins are at least two-fold significantly more abundant in the CSF of P15-21 Mpdz-/- mice (red circles, protein names are indicated) whereas only two proteins were more abundant in the CSF of Mpdz+/+ mice (green circles). The CSF composition was analyzed in 3 Mpdz-/- and 3 Mpdz+/+ mice. D. Images of 10 μm-thick sections from lateral ventricle CP villi of Mpdz+/+ and Mpdz-/- mice, immunolabeled for Nkcc1. The areas in the marked squares are magnified below. Scale bars, top, 50 μm, bottom 20 μm. The histograms show the mean fluorescence intensities per hCPEC in each cell group, normalized relative to the highest recorded intensity (mean ± SD, n=103). E. Immunoblots of LDLR and Mpdz, with β-actin as loading control. The MPDZ immunoblot shows the efficiency of the shRNA-mediated knockdown. Bands from three immunoblots were quantified by normalization to β-actin bands (mean ± SD). TABLES Table 1: Over-abundant proteins in the CSF of Mpdz-/- mice, and their parameters in the LC-MS/MS analysis, grouped functionally into 7 sets (from top to bottom): extracellular matrix, blood coagulation cascade, lipoproteins, immune response, cytokines and cytokine-binding proteins, enzymes and enzyme-binding proteins, and protease inhibitors. LFQ, label-free quantification. Gene ID MW (kDa) EXPANDED VIEW FIGURE LEGENDS The Expanded View material consists of 3 figures and one data set.
BRD4 directs hematopoietic stem cell development and modulates macrophage inflammatory responses (2019)
Figure 1

Figure1. Vav-Cre deletion of Brd4 blocks HSC development

(A) A schematic representation of targeted deletion of mouse Brd4. The exon-intron organization of the Brd4 locus (top). Targeting vector contained exon 3 flanked by LoxP sites (green) and a neomycin resistance gene flanked by FRT (white). Cre mediated recombination should cause deletion of exon 3 resulting in loss of BRD4 expression (bottom).

(B) Genotyping of Brd4fl/fl mice with ERT2-Cre (Cre) or without ERT2 -Cre (+/+). Tamoxifen treatment deleted 2.2 kb Brd4fl/fl fragment from Cre/+ and Cre/Cre mice (top). Cre/+, Cre/Cre and +/+ mice were confirmed by the presence of ERT2 -Cre band (Cre), or its absence (WT) (bottom).

(C) WT and Brd4 KO cells were tested for mRNA (left) and the protein (right) by qRT-PCR and immune blot assays. Values represent the average of three experiments +/- SD.

(D) Vav-cre mediated Brd4 depletion was verified for cells from Brd4fl/fl Vav-Cre mice (fetal liver or spleen) by qRT-PCR and Immunoblot (left and right). Values represent the average of three experiments +/- SD.

(E) Photographic images of E14.5 embryos from WT and KO mice.

(F) Corresponding Flow cytometric profile of Lin- fetal liver cells stained with C-Kit and Sca-1 antibodies examining early hematopoietic progenitor population (LSK) (left). LSK populations were further divided by the Flt3 marker to detect HSCs and MPPs (middle). The number of total cells, that of LSKs, HSCs, MPPs in a fetal liver were estimated on indicated days of embryonic development (E13.5, E14.5 and E17.5). Values are the average of 3-5 embryos +/- S.D (right).

(G) A schematic of hematopoietic stem cell and early progenitor cell pathways.

(H) Representative flow cytometric profile of CD71- cells stained for the myeloid and lymphoid markers. Values are the average of 3 E17.5 embryos +/- S.D.

Data information: Statistical significance in all figures was determined by two-tailed unpaired t test is indicated by: * p < 0.05, ** p < 0.01, *** p < 0.001, NS: no significance.
BRD4 directs hematopoietic stem cell development and modulates macrophage inflammatory responses (2019)
Figure 2

Figure 2. LysM-Cre deletion of Brd4 impairs development and proliferation of resident peritoneal macrophages

(A) Flow cytometry profiles of peritoneal cells from WT and KO mice stained for CD19 and F4/80 (left). FACS profile of CD19- subset showing CD11b+ and F4/80+ subsets of cells from WT and KO macrophage populations (middle). Cell numbers (right) represent the average of 4 WT and 4 Brd4 KO mice +/- S.D. Note a reduction in F4/80+ cells from Brd4 KO peritoneum.

(B) Flow cytometry profiles of peritoneal cells from IL-4C injected WT and KO mice (left). Cell numbers (right) represent the average of 4 WT and 4 Brd4 KO mice +/- S.D.

(C) Flow cytometry profiles of BrdU incorporation and Ki67, ARG1 expression in peritoneal cells (gated for F4/80+, CD19- macrophages) from IL-4C treated WT and KO mice (left). The numbers of cells positive for BrdU, Ki67 and ARG1 (right). The numbers represent the average of three experiments +/- S.D.

(D) Microarray analysis of macrophage gene expression in IL-4C treated WT or Brd4 KO mice. Macrophages were isolated from peritoneal cells by the MACS system. Differentially expressed genes were identified with a cut-off line of log 2fold change >2 difference with p value of < 0.05. Red dots indicate differentially expressed genes (KO vs WT) and gray dots are those below the cut-off line.

(E) Hierarchical clustering of differentially expressed genes. Representative downregulated genes in KO macrophages are shown by gene symbols. See GO analysis in Appendix Fig S2C. (F) qRT-PCR confirmation of representative genes downregulated in KO macrophages from IL-4C treated mice. Values represent the average of three independent experiments +/- S.D.

Data information: Statistical significance in all figures was determined by two-tailed unpaired t test is indicated by: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, NS: no significance.
BRD4 directs hematopoietic stem cell development and modulates macrophage inflammatory responses (2019)
Figure 3

Figure 3 Brd4 deletion partially inhibits LPS induced gene expression in BM-derived macrophages

(A) Experimental scheme for generating BM macrophages. BM cells from Brd4fl/fl ERT2Cre mice were cultured in the presence of M-CSF for 7 days and treated with LPS. Brd4 was deleted by Tamoxifen treatment (see Figure 1B for verification of Brd4 deletion).

(B) Flow cytometry profiles of CD11b+, F4/80+ macrophage population harvested on Day 7 from untreated (WT) or tamoxifen treated (KO) cultures (left). Histogram depicting F4/80 expression levels in WT and KO macrophages (middle). Total number of macrophage yields calculated from > 15 independent experiments. Values represent the average of 21 WT and 24 KO experiments +/- S.D.

(C) MA plots of RNA-seq data from untreated (UT) or LPS treated (4h) WT and KO macrophages. Differentially expressed genes were identified by a cut off line of log2FC>1 (FDR < 0.1).

(D) Venn diagram depicting the number of LPS induced genes in WT and KO macrophages. See explanations in the Figure for overlapped and nonoverlapped genes.

(E) qRT-PCR analysis of genes representative of the BRD4 independent group (unaffected by KO), dependent group (downregulated by KO) or upregulated by KO. Data were obtained from three independent macrophage samples. Values are the average of 3+/- S.D.

(F) GO terms of BRD4 dependent and independent genes, analyzed by the Enrichr program. Values in the overlap columns indicate the number of genes found in this study vs total number of genes in the GO terms.
BRD4 directs hematopoietic stem cell development and modulates macrophage inflammatory responses (2019)
Figure 4

Figure 4. LPS treatment increases BRD4 binding on BRD4 dependent and independent genes. (A) Genome-wide distribution of BRD4 in untreated (UT) and LPS treated macrophages. The numbers on the top represent total peak numbers.

(B) Heat maps of BRD4 occupancy on the promoter region (TSS +/- 5kb), aligned by the degree of BRD4 signal intensity in UT and LPS treated macrophages.

(C) Motif analysis of genome-wide BRD4 binding sites, assessed by the HOMER motif analysis algorithm.

(D) Immunoblot detection of BRD4 protein levels in UT and LPS treated WT macrophages. Forty μg of whole cell extracts from UT or LPS treated (1h or 4h) macrophages using antibody for BRD4 or β-actin (loading control).

(E) Binding of BRD4 and PolII and distribution of histone marks (H3K27ac and H3K9ac) on BRD4 dependent and independent genes (from -5kb of TSS to +5kb of transcription end site (TES)) in UT and LPS treated WT and KO macrophages. Normalized ChIP-seq signals (rpm/bp) are shown on the Y axis. Arrows show reduced signals in Brd4 KO macrophages.

(F) Gene tracks of normalized island-filtered ChIP-seq peaks for BRD4, Pol II, and indicated histone modification marks, along with RNA-seq peaks for a BRD4 independent gene (Ccl9) and a BRD4 dependent gene (Fcgr2b). Values on the Y axis are normalized ChIP-seq signals (rpm/bp).
BRD4 directs hematopoietic stem cell development and modulates macrophage inflammatory responses (2019)
Figure 5

Figure 5. Super-enhancers (SEs) in WT and KO macrophages

(A) BRD4 bound enhancers within 12.5kb were stitched together and ranked by BRD4 ChIP-seq signals (rpm) in untreated (UT, left) and LPS treated (middle) WT macrophages. The dotted line distinguished SEs from TEs. Gene names with the ranking in parenthesis are among genes closest to SEs. Venn diagram (right) showing nearest genes common in UT and LPS treated macrophages.

(B) Genome-wide gain and loss of BRD4 SEs upon LPS stimulation. Based on SE coordinates (rpm from LPS treated samples/rpm in untreated samples), changes greater or less than 2 folds (Log2) were plotted as gain or loss.

(C) Examples of nearest genes that gained or lost SEs upon LPS treatment. Gene tracks for gained and lost loci (near Tnf and Csf1r) showing clusters of BRD4, Pol II and H3K27ac signals. Red bar represents SE length.

(D) Meta-gene alignment of BRD4 signals on BRD4 ranked SEs. Pol II and H3K27ac ChIP-seq reads were aligned centering on BRD4 SEs of LPS treated WT macrophages.

(E) Enhancer plots obtained by the H3K27ac-ranking in LPS stimulated WT and KO macrophages.

(F) Venn diagrams showing genes nearest to H3K27ac SEs common in WT and KO macrophages (left), H3K27ac SE region from WT and KO macrophages (right).
BRD4 directs hematopoietic stem cell development and modulates macrophage inflammatory responses (2019)
Figure 6

Figure 6 Increased NF-κB binding in Brd4 KO macrophages

(A) Heat maps of p65 binding centered at the TSS on BRD4 enriched promoters.

(B) Meta-gene profile of p65 ChIP-seq signals (rpm/bp) from the TSS to TES and +/- 5kb on BRD4 independent genes (top) and BRD4 dependent genes (bottom).

(C) Gene tracks of BRD4 and p65 ChIP-seq peaks and RNA-seq profiles on a BRD4 independent gene (Il1b), a gene upregulated in Brd4KO macrophages (Ccl7), and a BRD4 dependent gene (Ptgs2).

(D) Enhancers ranked by increasing p65 signals in WT (left) and Brd4 KO macrophages (right).

(E) Meta-gene analysis of p65 occupancy on enhancers in WT (red) and KO macrophages (Blue).

Expanded View Figure Legends
Comparative proteomics reveals a diagnostic signature for pulmonary head-and-neck cancer metastasis (2018)
Figure 1

Figure 1. Genetic comparison of lung and head-and-neck carcinomas.

A Representative H&E and immunohistochemical stainings of CK5/6 and p63 in pulmonary and head-and-neck squamous-cell carcinomas. Scale bar: 500 µm.

B Heatmap showing somatic mutations in defined pathways and HPV status for all HNSCC and SQCLC cases analysed. (Blue, no mutation/ no HPV detected; red, mutation/HPV detected; white, no data available)

C Comparison of mutation rates in HNSCC (n=279) and SQCLC (n=178) cases from TCGA. Genes exhibiting a mutation rate >0.25 in both cancer types are labelled.
Comparative proteomics reveals a diagnostic signature for pulmonary head-and-neck cancer metastasis (2018)
Figure 3

Figure 3. Validation of proteomic differences of lung and head-and-neck carcinomas.

A Volcano plot adjusted p values for differential protein expression to averaged normalised SILAC ratios of two replicates. Red (higher expression in HNSCC) and blue (higher expression in SQCLC) dots indicate significantly regulated proteins (p < 0.05).

B Two-class comparison of genetic dependencies from a publically available genome-scale CRISPR-Cas9 screen of HNSCC (12 cell line) versus SQCLC (10 cell lines) identified a subset of differentially expressed proteins that were also differential dependencies in these tumor types. The x-axis represents the effect size of the mean difference of dependency scores in HNSCC compared to SQCLC cell lines. Positive effect size indicates a greater mean dependency in HNSCC; negative effect size indicates a greater mean dependency in SQCLC. The y-axis represents the statistical significance of differential enrichment calculated as -log10(p-value) from a two-sided t-test. The p-values used for this plot are uncorrected for multiple hypothesis testing. Highlighted in green are genetic dependencies that were also identified as differentially expressed proteins in our study.

C, D Immunohistochemical analysis of the expression of HMGCS-1 (p=0.0014),FTL (p=0.0001) (C),LGALS7 (p=0.0001) and FAM83H (p=0.0001)(D) in an independent cohort of 212 SQCLC and 343 HNSCC cases. Scale bar indicates 100µm. Shown are mean values and standard deviation. Statistical significance was assessed using Wilcoxon-Mann-Whitney test.

E Pathway-enrichment analysis for proteins differentially expressed in HNSCC and SQCLC.