EMBO/soda-nel huggingface dataset:
Single-cell transcriptomics reveals immune response of intestinal cell types to viral infection (2021)
Figure 1

Figure 1. Interferons protect human intestinal organoids from HAstV1 infection.

A. Cryo-sections of human organoids were analyzed for the presence of enterocytes (E-cad), Goblet cells (Muc-2) and tight junctions (ZO-1) by indirect immunofluorescence. Nuclei are stained with DAPI. Scale bar 25 μm.

B. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. 16hpi organoids were frozen, cryo-sectioned, and HAstV1 infected cells were visualized by indirect immunofluorescence (HAstV1 (red), nuclei (DAPI, blue). Scale bar 25 μm.

C. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. Organoids at 16 hpi were fixed and the presence of HAstV1 infected cells (green) were visualized by indirect immunofluorescence. Apical and basolateral membranes were immunostained for actin (magenta) and Laminin (red) respectively. Nuclei are stained with DAPI (blue). Scale bar is 20 μm.

D. Quantification of C with the percentage of infected cells determined.

E. Human intestinal organoids were infected with HAstV1. At indicated time post-infection the increase in viral copy number was determined by q-RT-PCR.

F. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. At 24 hpi the presence of IFNλ in the media was tested by ELISA. Dotted line indicates detection limit of the assay.

G. Same as E but for the induced levels of either type I IFN (IFNβ1) or type III IFN (IFNλ).

H. Human intestinal organoids were pre-treated for 24h with 2000IU/mL of IFNβ1 or 300ng/mL of IFNλ1-3. Interferons were maintained during the course of infection and the amount of HAstV1 copy numbers was assayed 24hpi by q-RT-PCR.

Data information: A-G Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.
Single-cell transcriptomics reveals immune response of intestinal cell types to viral infection (2021)
Figure 3

Figure 3. Single-cell profiling and multiplex in situ RNA hybridization of human ileum-derived organoids.

A. UMAP of scRNA-Seq data from human ileum-derived organoids (n=16,682 cells); dots corresponding to cells are colored by the cell type.

B. Dot plot of the top-three marker genes for each cell type. The dot size represents the percentage of cells expressing the gene; the color represents average expression across the cell type. Fig.S3B shows an extended list of the marker genes. All cell-type-defining marker genes are provided in Dataset EV2.

C. Representative images showing multiplex in situ RNA hybridization of marker genes in an organoid. DAPI in blue.

D-I. Visualizations for cell-type markers for the major intestinal cell types, showing single-cell expression intensities of the cell-type markers on the UMAP (left) and multiplex in situ RNA hybridization; red corresponds to viral RNA and a green corresponds to cell type markers (right).

J-M. Percentage of stem cells (OLFM4) goblet cell (FGCBP), mature enterocytes (APOA4) and cells of the enterocyte lineage (FGCBP) from the total organoid cells population (black bar) in mock-treated organoids and after HAstV1 infection. The percentage of infected cells (red bars) and bystander cells (blue bars) within each cell type is shown. The data was obtained from multiplex in situ RNA hybridization in 2D organoids. n=10, mean ± SEM. Statistics show comparison of infected cells between mock-treated, 4h pi and 16h pi organoids. An Ordinary one-way ANOVA and Tukey's multiple comparisons test was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001.
Single-cell transcriptomics reveals immune response of intestinal cell types to viral infection (2021)
Figure 5

Figure 5. Multiplex in situ RNA FISH visualizes immune signature in HastV1-infected and bystander cells.

A. One image per condition was used to show the multiplex in situ RNA FISH. A representative region of the images was chosen and cropped in order to have a zoom-in of a specific area for better visualization. Shown are the enterocyte lineage marker FABP6 (green), HAstV1 infection (white), and CCL2 gene expression (red) of mock-treated and infected organoids. DAPI is in blue. White arrows show co-localization of FABP6 and CCL2 with HAstV1 signal. Scale bar 100µm.

B-D. Fluorescence intensity (arbitrary units) of innate immune marker expression in stem cells (OLFM4 positive), goblet cells (FCGBP positive), enterocyte lineage cells (FABP6 positive), and mature enterocytes (APOA4 positive). Bystander cells are in blue and infected cells in red. 10-90 percentile box plots, each dot represents one cell (mock N=2274, 4h N=3682, 16h N=1510. C. mock N=2693, 4h N=3706, 16h N=1921. D. mock N=2274, 4h N=3706, 16h N=1514.) . Blue statistics show a comparison of bystander cells between cell lineages and red statistics show the comparison of infected cells between cell lineages. Ordinary one-way ANOVA and Tukey's multiple comparisons test were used. Grey statistics show the comparison between infected and bystander cells within each cell lineage. Unpaired t-test with Welch's correction was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Extended View Figure Legends
Spt5 histone binding activity preserves chromatin during transcription by RNA polymerase II (2022)
Figure 1

Figure 1

Eukaryotic Spt5N binds histones H3-H4 via an evolutionarily conserved motif. (A) Domain structure of Spt5 / NusG proteins. The enlarged inset shows a conserved region of Spt5N from Saccharomyces cerevisiae (S.c.), Schizosaccharomyces pombe (S.p.), Aspergillus nidulans (A.n.), Homo sapiens (H.s.), Xenopus laevis (X.l.) and Arabidopsis thaliana (A.t.). Spt5N contains three invariant residues that were mutated in Spt5-3A (indicated by red asterisks), whilst residues with conservative substitutions are denoted by blue asterisks. (B) GST, GST-MCM2N (human) and GST-Spt5N (yeast) were mixed as indicated with yeast histones H3-H4, in the presence of glutathione agarose beads (see STAR method for further details). The input and pulldown samples were analysed by SDS-PAGE before staining with colloidal Coomassie blue. The asterisks denote truncations of GST-Spt5N. (C) Similar experiment to (B) but with histones H2A-H2B. (D) Similar experiment to (B) to compare histone binding by GST-MCM2N and GST-MCM2N-2A (with Y81A Y90A mutations of conserved residues). (E) Analogous experiment to (D), comparing histone binding by GST-Spt5 and GST-Spt5N-3A (with F180A E184A V187A mutations of conserved residues). (F) Budding yeast Spt5N and Spt5N-3A were expressed as fusions to Protein A in budding yeast cells. Cell extracts were treated with DNase as indicated, to release nucleosomal histone complexes from chromatin, before immunoprecipitation (IP) of Spt5N / Spt5N-3A on magnetic beads coated with IgG. The bound proteins were eluted and then analysed by mass spectrometry (the panel indicates the detected spectral counts). (G) Samples from an analogous experiment to that shown in (F) were analysed by immunoblotting of the indicated factors. The asterisk indicates a non-specific band in the immunoblot for histone H2B. (H). Budding yeast cells expressing fusions of Spt5N and Spt5N-3A to Protein A were grown in parallel with cells expressing the equivalent wild type and mutant versions of fission yeast Spt5N (Sp Spt5N and Sp Spt5N-3A - the latter contained the F146A, E150A and V153A mutations). See also Figure EV1.
Spt5 histone binding activity preserves chromatin during transcription by RNA polymerase II (2022)
Figure 2

Figure 2

Mutation of the histone-binding motif of Spt5N is lethal, even in the presence of wild type Spt5. (A) Linearised versions of the indicated plasmids were transformed into SPT5-9MYC budding yeast cells and integrated at the ura3 locus. For each transformation, four colonies were analysed by immunoblotting and DNA sequencing as shown. Colonies transformed with plasmid expressing Spt5-3A either lost the plasmid (1), contained the SPT5-3A DNA but failed to express Spt5-3A protein (2+4) or experienced a gene conversion and thus expressed wild type Spt5 from the integrated plasmid DNA (3). (B) Serial dilutions of yeast cells expressing the indicated fragments of wild type Spt5 or Spt5-3A from the GAL1,10 promoter were grown on the indicated medium, together with a control of wild type cells. (C) Yeast cells were generated that expressed Protein A-tagged versions of wild type Spt5 (full length or the indicated truncations), or full length Spt5-3A, under the control of the GAL1,10 promoter. Cells were grown in medium containing galactose and the ProteinA-tagged Spt5 proteins were isolated from cell extracts on magnetic beads coated with IgG. Association of the tagged Spt5 proteins with the elongating form of Pol II was monitored by immunoblotting, using antibodies specific to the indicated phosphorylation sites in the C-Terminal Domain repeats of the Rpb1 subunit of Pol II. (D) Control and spt5-AID strains were grown at 30°C in rich medium, before addition of auxin (0.5 mM 3-indoleacetic acid or IAA) for 1 hour. Spt5 protein was monitored in cell extracts by immunoblotting. (E) Serial dilutions of control cells or spt5-AID cells, expressing the indicated versions of Spt5 from the GAL1,10 promoter, were grown on the indicated medium.
Spt5 histone binding activity preserves chromatin during transcription by RNA polymerase II (2022)
Figure 3

Figure 3

The histone-binding activity of Spt5N is dispensable for progression of the Pol II elongation complex through transcription units. (A) Experimental procedure, based on the yeast strains YCE281, YCE350 and YCE356. (B) Immunoblot analysis at the indicated times for the samples described in (A). (C) Cells from the end of the experiment described in (A) were fixed and processed for ChIP-Seq, using antibodies specific for phosphorylated Serine 5 in the C-Terminal Domain of the Pol II catalytic subunit Rpb1 (CTD-Ser5P). For each of the three indicated strains, the histograms represent the normalized read density (DNA sequence read count per million reads, or RPM), across a sample gene body. (D) Average of the normalised read density (RPM) for the CTD-Ser5P ChIP-Seq data from two independent experiments, for regions spanning 1kb upstream and downstream of the Transcription Start Site (TSS) of actively transcribed genes across the yeast genome (the analysis focussed on 821 actively transcribed genes, as described in Materials and Methods). (E) For each transcribed gene, an RNA Pol II Traveling ratio was calculated as indicated. (F) Log2 values of the calculated Pol II traveling ratios for each actively transcribed gene were arranged in order from low to high and then plotted against percentile. (G) Principal component analysis of two independent experiments, for the Pol II CTD-Ser5P ChIP-Seq signal across the yeast genome in the three indicated strains.
Spt5 histone binding activity preserves chromatin during transcription by RNA polymerase II (2022)
Figure 4

Figure 4

Spt5N histone binding activity preserves nucleosome integrity during transcription. (A) Samples from the experiment described in Figure 3A were also processed for MNase-Seq. For each of the three indicated strains, the histograms represent the normalized DNA sequence read count per million reads (RPM), across a sample gene body. (B) Average of the normalised read density (RPM) for the MNase-Seq data from two independent experiments, for regions spanning 1kb upstream and downstream of the Transcription Start Site (TSS) of actively transcribed genes across the yeast genome (as above, the analysis focussed on 821 active genes, as described in Materials and Methods). (C) Samples from the same experiment were further processed for ChIP-Seq using antibodies to histone H3K4me3. For each of the three indicated strains, the normalised read count per million reads is presented across a sample gene body. (D) Average of the normalised read density (RPM) for the H3K4me3 ChIP-Seq datasets from two independent experiments, for regions spanning 1kb upstream and downstream of the Transcription Start Site (TSS) of actively transcribed genes. (E) Principal component analysis of two independent experiments, for the H3K4me3 ChIP-Seq signals across the yeast genome in the three indicated strains. (F) Ratio of the H3K4me3 ChIP-Seq signal (TSS to +500 bp) in cells expressing Spt5-3A and cells expressing wild type Spt5. The boxes extend from the 25th to 75th percentiles whilst the 'whiskers' illustrate the minimum and maximal values and the lines within each box correspond to the median values. The data are expressed as a function of the gene expression level, based on the read density of the ChIP-Seq data for CTD-Ser5P (from TSS to TTS) in Figure 3, ordered from lowest to highest values in four quartiles (colour coded as indicated). The correlation between H3K4me3 in cells expressing Spt5-3A (spt5-AID GAL-SPT5-3A) and control cells (spt5-AID GAL-SPT5) is progressively worse from Q1 to Q4, for genes expressed at progressively higher levels.
Endothelial cell-derived Angiopoietin-2 is a therapeutic target in treatment-naive and Bevacizumab-resistant glioblastoma (2015)
Figure 1

Figure 1: Endothelial Ang-2 upregulation correlates with WHO grading in human gliomas. ELISA displaying human Ang-2 (A) and Ang-1 (B) level in serum of healthy patients (Ang-2 n=4; Ang-1 n=21) or patients with low grade glioma (WHOII) (n=5), anaplastic astrocytoma (WHOIII) (n=7) or glioblastoma (WHOIV) (n=39) are shown. The TCGA database was accessed to obtain gene expression level for Ang-1, Ang-2, Tie2 and Tie1 in GBM in comparison to normal brain (C). Ang-2 expression and quantitative analysis of Ang-2 in healthy human brain (n=3), low grade diffuse glioma (n=14), anaplastic astrocytoma (n=12) or glioblastoma (n=11) is shown (D). Co-staining of Ang-2 and vWF (endothelial cells), αSMA (mural cells) and IBA1 (microglia) in different glioblastoma specimen. Normal brain tissue was used to assess Ang-2 staining specificity (E). Ang-2 predicts survival of glioblastoma patients (F). If not indicated differently, Kruskall-Wallis (Dunn´s post test) was applied, ** p < 0.05, *** p < 0.005; data are mean ± SEM
Endothelial cell-derived Angiopoietin-2 is a therapeutic target in treatment-naive and Bevacizumab-resistant glioblastoma (2015)
Figure 2

Figure 2: Endothelial Ang-2 expression reduced pericyte coverage and increased macrophage infiltration in experimental glioblastoma. GL261 cells were intracerebrally transplanted in wild type or Ang-2 gain-of-function mice (Ang-2 DT). Brain tumor sections were stained with antibodies against CD31 and desmin (A), and microvessel densities (MVD) and pericyte coverage were determined (WT n=14; Ang-2 DT n=6) (B). FACS analysis of infiltrating macrophages in Ang-2 DT normal brain (w/o tumor) compared to WT is shown (C, dot plot; D, quantification) (WT n=5; Ang-2 DT n=3). Immunohistochemistry staining for monocytes/macrophages (F4/80) and neutrophils (Ly6G; arrowheads) in brain tumors derived from WT or Ang-2 overexpressing mice (E), Quantitative analysis of C (F) (WT n=14; Ang-2 DT n=10). Kaplan-Meier survival curve of GL261 tumor-bearing WT and Ang-2 DT mice (WT n=17; Ang-2 DT n=14) (E). Statistical analyses were performed using an unpaired Student´s t-test (B, F), one-way Anova (Tukey post test) (D), Log-rank and Wilcoxon (G) * p < 0.05, ** p < 0.01; data are mean ± SEM
Endothelial cell-derived Angiopoietin-2 is a therapeutic target in treatment-naive and Bevacizumab-resistant glioblastoma (2015)
Figure 3

Figure 3: Infiltrating leukocyte subsets in human glioma. Paraffin sections of healthy human brain (n=3), low grade glioma (n=12), anaplastic astrocytoma (n=7) and glioblastoma (n=9) were stained for IBA1 (macrophages/microglia), CD15 (granulocytes) and CD3 (T-cells) (A). Quantification of leukocyte subsets in human glioma specimens (B). For statistical analysis Kruskall-Wallis (Dunn´s post test) was applied.* p < 0.05, ** p < 0.01; data are mean ± SEM
Endothelial cell-derived Angiopoietin-2 is a therapeutic target in treatment-naive and Bevacizumab-resistant glioblastoma (2015)
Figure 4

Figure 4: Dual anti-Ang-2 and anti-VEGF therapy acts synergistic on vascular normalization and macrophage infiltration in experimental GBM. Immunofluorescence staining of CD31 and desmin in GL261 glioblastoma after single and dual treatment with anti-Ang-2 (AMG386) and anti-VEGF (aflibercept) (A). Corresponding quantitative analysis of microvessel densities and pericytes numbers (B; control n=24; AMG386 n=10; aflibercept n=12; AMG386 + aflibercept n=13). Staining for mouse IgG (red) as a surrogate parameter for vascular leakage and CD31 (green) is shown in GL261 glioblastoma sections following different anti-angiogenic treatments (C) with quantification in (D; n=3). Quantification of necrotic areas in GL261 glioblastoma sections after anti-angiogenic treatment is shown as percent of whole tumor area (E; control, aflibercept, AMG386 + aflibercept n=4; AMG386 n=3). Analysis of Glut1 immunoreactivity in GL261 glioblastoma sections (F; n=3). Double-immunofluorescence stainings of macrophages (F4/80) and tumor vessels (vWF) in mouse GBM after treatment with AMG386, aflibercept or the combination of both are shown in (G). Quantitatve analysis of tumor infiltrating leukocytes (F4/80+, CD3+, Ly-6G+) following anti-angiogenic treatment is displayed in (H; control n=28; AMG386 n=12; aflibercept n=11; AMG386 + aflibercept n=12). Kaplan-Meier survival analysis of GL261 tumors grown in C57BL/6 mice following anti-angiogenic treatment (I; control n=38; AMG386 n=13; aflibercept n=12; AMG386 + aflibercept n=13). Double-immunofluorescence stainings with anti-F4/80 and anti-CD206 in brain tumor sections of mice treated with anti-Ang-2 (AMG386), anti-VEGF (aflibercept) or the combination of both are shown in (J). Corresponding quantitative analyses of tumor infiltrating F4/80+ cells and CD206+ cells, and the ratio of CD206+ vs. F4/80+ upon anti-angiogenic therapy are displayed in (K; control n=21; AMG386 n=13; aflibercept n=9; AMG386 + aflibercept n=4). Flow cytometry of tumor infiltrating macrophages following enzymatic dissociation of mouse GL261 brain tumors plus/minus anti-angiogenic therapy. Percent of CD206+ (L) and MHC class IIHI cells (M) among CD45+CD11b+GR1-F4/80+ cells are displayed (L,M; control n=4; AMG386 n=4; aflibercept n=5; AMG386 + aflibercept n=5). Statistical analyses were performed using one-way ANOVA and Tukey’s multiple comparison except for Log-rank and Wilcoxon (I) and Kruskall Wallis (B). * p < 0.05, ** p < 0.01, *** p < 0.005, # p < 0.005 of aflibercept + AMG386 vs aflibercept; data are mean ± SEM (A-K)
Endothelial cell-derived Angiopoietin-2 is a therapeutic target in treatment-naive and Bevacizumab-resistant glioblastoma (2015)
Figure 5

Figure 5: Anti-VEGF therapy led to decreased infiltration of CD68+ macrophages in human GBM. Anti-CD68 (A), anti-CSFR1 (B) and anti-CD206 (C) immunohistochemistry of patient samples derived from treatment-naive GBM (n=24), post-radiochemotherapy (S/RTx/CTx) (n=7) and post-radiochemotherapy + bevacizumab (S/RTx/CTx/Bev) (n=29) therapy is shown. Corresponding quantification of tumor infiltrating cells is displayed (anti-CD68, D), (anti-CSF1R, E) and (anti-CD206, F). Ratio of CD206+ vs. CD68+ cells (G). Amount of CD206+ perivascular cells per blood vessel (H). Double staining of Ang-2 (brown) and CD206 (red) of a S / RTx / CTx / Bev patient, arrowheads indicating perivascular CD206+ cells around Ang-2+ vessels (I). Arrows pointing on parenchymal CD206+ cells (I). CD206+ cells in relation to the percentage of hypoxic (CAIX positive) tumor area (J). Statistical analysis: Kruskall-Wallis (Dunn´s post test). * p < 0.05, ** p < 0.01; data are mean ± SEM
Endothelial cell-derived Angiopoietin-2 is a therapeutic target in treatment-naive and Bevacizumab-resistant glioblastoma (2015)
Figure 6

Figure 6: Bevacizumab therapy leads to reduced vessel density and increased Ang-2 expression. Immunohistochemistry stainings with antibodies directed against CD31 (A) and Ang-2 (B) in treatment-naive GBM, post-radiochemotherapy (S/RTx/CTx) and post-radiochemotherapy + bevacizumab (S/RTx/CTx/Bev) therapy are displayed. Quantitative analyses of microvessel densities and Ang-2+ vessels are shown in (C) and (D). The ratio of Ang-2+ vs. CD31+ vessels was determined in (E). Western Blot and corresponding quantification of CD31 and Ang-2 in control and aflibercept treated mice (F). CAIX staining of treatment-naive GBM and S / RTx / CTx / Bev patients (G). Analyses of hypoxic (CAIX+) area with regard to the whole tumor in human GBM (H). Tumor-infiltrating lymphocytes (CD8/CD3) in human patients (I). Kruskall-Wallis (Dunn´s post test). * p < 0.05, *** p < 0.005; treatment-naive GBM n=26, S/RTx/CTx n=7, S/RTx/CTx/Bev n=29; data are mean ± SEM
CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion (2018)
Figure 3

Figure 3. Multiple PAM influence on binding to a target site

DNA and crRNA sequence used for experiments of a single target and multiple PAMs. DNA sequence is shown for the single-target single-PAM construct and schematic representation is shown for multiple-PAM containing constructs.

FRET histogram of all binding events from the construct containing a single PAM next to the partial target. Y axis represents total number of counts.

Example FRET traces from single-target multiple-PAM experiments. Top trace shows an example of a single-step binding event and a binding event that starts at a lower FRET state before transitioning to productive binding high-FRET state. Bottom trace shows an example of FRET fluctuations between the high-FRET state and lower FRET states.

A histogram showing the percentage of dynamic. The percentage values were obtained from five experiments on five different days. Error bars represent standard error of the mean. The percentage of events showing dynamic binding behavior was calculated by dividing the number of binding events that showed such behavior by the total number of binding events.

A scatter plot showing the time Cas9 spends in a lower FRET state before transitioning to a high-FRET state. The dwelltimes increase with the number of PAMs adjacent to the target. Such dwelltime could not be determined for a construct containing a single PAM. The values were obtained from five experiments on five different days. Error bars represent standard error of the mean.
CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion (2018)
Figure 4

Figure 4. Mechanism of lateral diffusion

DNA sequences used in tandem target experiments. Complementary sequences to crRNA are marked in pink boxes and PAMs are marked in green boxes. Distances between the protospacers are indicated above the sequences. An illustration of Cas9 binding to each target site resulting in either high FRET (target H) or low FRET (target L) is shown on the right.

Example fluorescence traces showing Cas9 transitioning between H and L targets with increasing target distances (right). FRET histograms constructed from events that show fluctuations showing two FRET peaks corresponding to binding to either target. High FRET peak stays the same in each histogram, but lower FRET peak moves to lower FRET values as the distance between targets increases.

A scatter plot showing the probability of Cas9 transitioning between targets before dissociation. The values are averages from five measurements and error bars represent standard error of the mean. The transition probability (p = T / (T+D)) was calculated by summing up all the number of FRET transitions (T) and dividing T by the sum of T and the total number of dissociation events (D).

A histogram showing all dwelltimes from single-target controls and tandem-target experiments. τ1 is similar for single-target control and tandem target experiments. τ2 only appears in tandem target experiments and is over 20-fold higher than τ1 or τst. The values represent the mean dwelltimes that were obtained by fitting dwelltime distributions by either single or double exponential decay function from five measurements. Error bars represent standard error of the mean.