EMBO/soda-nel huggingface dataset:
Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor (2019)
Figure 6

Figure 6. The engineered flux-sensor reports glycolytic flux with a high dynamic flux range. (A) Overview about the different design steps in our promotor engineering strategy (cf. also Appendix Figure S4). (B) The four reporter plasmids were transferred to the wildtype strain containing the CggR (R250A) under the control of the PTEFmut7. The strength of the four promoters was assessed by quantifying YFP (FL1) and mCherry (FL3) fluorescence in exponentially growing wildtype cells in minimal medium with 10 g l-1 glucose. The FL1 and FL3 fluorescence shown is the non-background corrected median of 100 000 cell events. The non-FL control is the signal from a wildtype strain grown under the same conditions. (C) The background fluorescence, assessed by the wildtype harboring the YCplac33 plasmid, was substracted from FL1 and FL3. The final reporter activity is the ratio of the background-corrected YFP and mCherry values. Error bars represent the standard deviation of three independently determined ratios from three replicate experiments. (D,E) Reporter activity of the sensor across (D) multiple FBP levels and (E) glycolytic fluxes. The glycolytic flux is reported as the flux between the metabolites fructose 6-phosphate (F6P) and fructose-1,6-bisphosphate (FBP). Glycolytic fluxes were here estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al, 2019). Reporter activity is given by the YFP/mCherry ratio, calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry. Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid (Appendix Table S8). The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR. Error bars represent the standard deviation of at least three replicate experiments. (F) Fraction of CggR bound to FBP across FBP concentrations. The red arrows indicate the shift in the percentage of CggR bound to FBP achieved in the R250A variant. The percentage of CggR molecules bound to FBP was calculated after normalizing the Tm values for unbound/bound state using the Tm at 0 mM FBP as unbound and at 36 mM (corresponding to maximum FBP concentration used) as total bound states. The curve fitting of the normalized values of CggR fraction bound to FBP was performed using a one-site specific binding model in GraphPad. The solid line corresponds to the wildtype CggR and the dashed line to the R250A variant. Vertical lines delimit the physiological FBP range.
Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation (2020)
Figure 1

Figure 1: Bulging cells divide to form the monoclonal core of the IAHC (A) Schematic representation of initial events of IAHC formation. (i) A single cell protrudes into the lumen of the aorta and starts expressing CD41. (ii) The CD41:YFP+ bulging cell undergoes mitosis (iii), forming the core of the IAHC. (B) Expression of Kit (magenta) largely overlaps with the expression of CD41:YFP (yellow) in IAHC. Multistack reconstruction of confocal images. Scale bars: 30μm overview, 10μm magnification. (C) Snapshots of Movie EV1. Time-lapse of embryonic organotypic slice culture from CD41:YFP (yellow): H2B-GFP (blue) reporter mouse shows the emergence of a single cell protruding from the endothelial layer into the aortic lumen. Scale bars: 50μm (overview), 10μm (magnification). Time expressed in hh:mm:ss. (D) Snapshots of Movie EV2. Time-lapse of embryonic organotypic slice culture from CD41:YFP (yellow): H2B-GFP (blue) reporter mouse shows a single CD41:YFP expressing cell that divides once on the aortic endothelium. Scale bars: 100μm (overview), 20μm (magnification). Time expressed in hh:mm:ss. (E) BrdU incorporation in the Kit+ IAHC upon cumulative administration. Multistack reconstruction of confocal images. Representative picture of a short BrdU pulse (1h). Scale bars: 50μm (overview), 10μm (magnification). (F) Quantification of the percentage of Kit+ cells within IAHCs incorporating BrdU using confocal images. All cells (Labelling Index=100%) can be labelled after 8h, indicating the lack of quiescent cells inside the IAHCs. Graph represents mean±SE.
Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation (2020)
Figure 2

Figure 2: IAHCs have a monoclonal, proliferative origin but become polyclonal at later stage. (A) Schematic representation of time of induction of recombination in VeCadCreER:Confetti pregnant females and analysis of embryos. Tamoxifen was injected before cluster initiation (E7.5 or E8.5) and embryos collected at the peak of cluster formation (E10.5 or E11.5). (B) Quantification of recombination efficiency at E10.5 and E11.5 using confocal images. Bars represents the mean±SD of the percentage of cells expressing single colored cassettes in CD31+ endothelial cells (n=2). (C,D) Transversal section of a recombined VeCadCreER:Confetti AGM from an E10.5 embryo and immunostained with CD31 (C) (white in image, black in label) or Kit (D) (white in image, black in label). Multistack reconstruction of confocal images. Scale bars 30μm (c) and 20 μm (D). (E) Schematic representation of possible outcomes in the IAHC after recombination: Clusters derived from sequential division of one colored cell will be unicolored (left panel). Clusters will be multicolored (right panel) when derived from several, independently recombined endothelial cells. (F) Analyses of recombination in 40 clusters. Bars representing single clusters are grouped based on the number of Kit+ cells. Recombination induction follows the schema in the black square. (n=12 embryo). (G) Representative confetti recombination on clusters of different sizes. Small cluster are monoclonal (left panel), while larger clusters are polyclonal (right panel). Inlet shows relevant confetti channel. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bars 5 μm.
Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation (2020)
Figure 3

Figure 3: Dll4-ligand expression in the IAHC varies with cluster size (A) Detail images of Dll4 staining in Kit+ cells of IAHC of different sizes. Dll4 expression inversely correlates with the size of the cluster: 2-5 cell IAHCs are uniformly Dll4-positive (upper and middle panel), >5 cell IAHCs are partially Dll4 positive (lower panel). Multistack reconstruction of confocal images. Scale bars: 10 μm. (B) Quantification of Kit+ Dll4+ cells using confocal images. Bars represent the percentage of Dll4 expressing cells within a cluster in each category of cluster size. Mean±SD (total 17 clusters analysed, n=2). (C) Overview of Dll4-ligand expression in the embryonic aorta at E10.5. Dll4 is expressed in the majority of the endothelial layer. Scale bar: 30μm
Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation (2020)
Figure 4

Figure 4: Blockage of Dll4 increases the number of cells in IAHCs (A)Whole mount images of the aorta endothelium stained for CD31 (white in image, black in label) and Kit (IAHC, cyan) upon IgG (left) or αDll4 antibody treatment (right). Multistack reconstruction of confocal images. Red arrows indicate IAHCs on the ventral part of the aortic wall. Asterisks indicate aspecific autofluorescence. Scale bar: 50μm. (B) Representative IAHCs of IgG and αDll4 treated embryos. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Note the increased IAHC size in the right panel. Scale bars: 50μm (overview), and 10μm (magnification). (C) Frequency of IAHC categories based on the number of Kit+ cells forming a cluster. Quantification of confocal images. Bars represent the mean±SE (n=6) (D) Number of Kit+ cells per IAHC increases following αDll4 antibody treatment. Each dot represents one cluster. Quantification on confocal images. Statistical analysis: Mann-Whitney U test. ****p< 0.0001. (E) Total number of IAHCs per 100μm. Quantification of confocal images. Mean±SE. Statistical analysis: t-test. ns p> 0.05; (n=6). (F) Quantification of the number of Kit+ cells per 100 μm using confocal images. Bars represent the mean±SE. Statistical analysis: t-test. ns p> 0.05; ***p< 0.001 (n=6).
Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation (2020)
Figure 5

Figure 5: changes in gene expression and decrease in Notch activity after Dll4 blockage. (A) Experimental design: E10.5 embryos were injected with IgG or αDll4, incubated for 5h and then dissected. The indicated IAHC subpopulations (kit+CD45+; kit+CD45-;kit-CD45-CD41+ were purified and RNA was sequenced. (B) PCA of the different subpopulations from IgG or αDll4 treated embryos based on normalized gene counts.(C) PCA considering the DEG according to the treatment specific for each subpopulation. (D) Function enrichment analysis of DEG in the Kit+CD45- population according to the treatment(E) Experimental design and sorting gate for Kit+CD45- cells for single cell RNAseq (n=483 cells).(F-G) tSNE distribution of IgG and αDll4-treated Kit+CD45- cells from single cell RNA-seq data. (F) cells corresponding to each treatment are indicated and (G) different clusters identified by ICGS (F) are represented.1-3 are endothelial-like cells and 6-7 are hematopoietic-like cells.(H) Violin plots of Notch family elements expression in IgG and αDll4 treated cells (adj p val: *<0.1 and *** p<0.00001). (I) Percentage of cells in each cell cycle phase extracted from single cell RNA-seq analysis. Bars represent the average±SE (n=483).
Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation (2020)
Figure 6

Figure 6: Cell recruitment is increased in Dll4-blocked IAHCs

(A) Bar graph represents the frequency of IAHCs containing recombined cells per embryo under the indicated conditions. Quantification of confocal images. Mean±SE (n=15 for untreated, n=7 for IgG and αDll4 treated). (B) Representative images of recombined IAHCs following IgG or αDll4 conditions. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bar: 5μm. (C) Quantification and classification of IAHC containing recombined cells in TAM-induced VeCadCreER:Confetti embryos after IgG or αDll4 treatment. Embryos treated with αDll4 contain more IAHC with colored cells. Quantification of confocal images (n=7 embryos). (D) Representative images of recombined endothelial cells in transversal sections of the aorta. The distribution and amount of recombined endothelial cells is similar in IgG and αDll4 conditions. CD31 is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bar: 50μm. (E) Quantification of recombined cells in the aortic endothelium using confocal images. Mean±SE (n=3).
Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation (2020)
Figure 7

Figure 7: Mathematical model supports a role for Dll4 in controlling cell recruitment into IAHCs. (A) Schematics of the probabilistic model of IAHC formation. The processes considered in the model are (1) initiation of cluster, (2) cell division event, (3) recruitment event, and (4) cluster detachment. Each process in the model is described as a typical time scale (Tcluster lifetime, Tcell division, Trecruitment). In case of αDll4 treatment, the final five hours of the simulation are assumed to have a different recruitment time. See details of the simulation in the methods. (B) Heat map showing the probability (or p-value) that the simulation produces mean cluster size and fraction of colored cells that match those observed experimentally for the IgG treatment. This probability is calculated for different values of cluster lifetimes and mean recruitment times (values in x and y). 4000 simulations were performed for each set of parameters (each point in the heat map). The most likely parameter values (yellow) correspond to a cluster lifetime of 21 hours and mean recruitment time of 11 hours. Red dashed line indicates the cross-section with the most likely cluster lifetime (21 hours). (C) A graph showing the probability that the simulation produces mean cluster size and fraction of colored cells that match those observed experimentally for the αDll4 treated clusters (blue line). Here, the cluster life time is taken to be 21 hours (taken from b) and the probability to match the experimental data is calculated for different recruitment times in the final 5 hours of the simulation (x-axis). The most likely mean recruitment time for the αDll4 treatment is 5 hours (peak of blue line). For comparison, the most likely mean recruitment time for the IgG treatment is 11 hours (Red dashed line, corresponding to the red dashed in (B)). Hotelling's T-Squared test. (D) Examples of simulations of IAHC distributions for the IgG (left) or αDll4 (right) treated samples. Simulations were performed with the most likely parameter values described in b and c. Note that only clusters that have at least one colored cell are shown (i.e clusters with non-colored cells are not represented).(E) Snapshots of Movie EV5. Time Lapse of embryonic organotypic slice from a CD41:YFP: H2B-GFP reporter mouse showing the recruitment of CD41:YFP negative cells (magenta central point-tracking analyses) migrating towards the IAHC (CD41:YFP+ cells). Green central point and relative tracking identify cells with non-directional movement. Scale bar: 5μm. Time expressed in hh:mm:ss.
Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation (2020)
Figure 8

Figure 8: Interference with the Dll4 ligand functionality results in increased hematopoietic potential in embryonic AGM. (A) Schematic representation of functional assays for testing the hematopoietic potential of AGM. Embryos were injected intracardiacally and incubated for five hours with αDll4 or IgG control. Explant cultures were either exposed to the relevant treatment for 5h before FACS analyses or incubated for 48h before transplantation into irradiated mice. (B) Colony-forming assays using E10.5 AGMs derived from embryos treated with IgG or αDll4. Mean±SD (n=3) No significant differences were detected (C-E) FACS analyses of Gfi1+ cells derived from explants treated with IgG or αDll4 for 5 hours (C). Analyses of the hemogenic Gfi1+Kit-(D) and the Gfi1+Kit+ (E) within the CD31+CD45- population. Statistical analysis: t-test. Box plots show upper and lower quartile; center line represents mean (n=4). *p< 0.05; **p< 0.005; non significant comparisons are not indicated (F) Percentage of reconstitution (chimerism determined by CD45.1 and CD45.2 expression) in peripheral blood (PB). Embryos treated with IgG or αDll4 were kept in explant culture for 48hours and then transplanted into irradiated recipients. Peripheral blood was analyzed at different time points. Each dot/square represents a single transplanted mouse. (G) Analysis of progenitors (lin-sca1+kit+) and HSCs (Lin-sca1+kit+CD48-CD150+) in the donor population in the bone marrow after 4 months of transplantation. (H) Model for recruitment of Gfi1+ cells into the IAHC. Recruitment of Gfi1+endothelial cells increases upon Dll4 blockage.

EXPANDED VIEW LEGENDS
ST3GAL5-catalysed gangliosides inhibit TGF-β-induced epithelial-mesenchymal transition via TβRI degradation (2022)
Figure 1

Figure 1. TGF-β decreases the abundance of glycosphingolipid (GSL)-glycans.

A, B Average copy numbers of (A) total GSL-glycans and (B) individual GSL-glycans per cell in NMuMG cells stimulated with TGF-β.

C, D Average copy numbers of (C) total GSL-glycans and (D) individual GSL-glycans per cell in A549-VIM-RFP cells treated with TGF-β.

GSL biosynthesis scheme. GSLs present in both NMuMG cells and A549-VIM-RFP cells are highlighted by red boxes.

F, G Immunoblot analysis of ST3GAL5 in (F) NMuMG and (G) A549-VIM-RFP cells treated with vehicle control or TGF-β for the indicated time. Vinculin and GAPDH: loading controls.

Data information: Unless stated otherwise, TGF-β (2.5 ng/mL) and vehicle control were applied for 48 h. The histograms show the mean ± SD values from three biological replicates (n=3). *, P ≤ 0.05; **, P < 0.01 based on unpaired Student's t test. GT1b: internal normalization control.
ST3GAL5-catalysed gangliosides inhibit TGF-β-induced epithelial-mesenchymal transition via TβRI degradation (2022)
Figure 2

Figure 2. KO of Ugcg, a key enzyme in the biosynthesis of GSLs, promotes TGF-β signaling and TGF-β-induced EMT in NMuMG cells.

Combined extracted ion chromatography (EIC) of GSL-glycans in Ugcg KO and control NMuMG cells. GT1b: internal normalization control.

Mouse TGF-β gene signatures were enriched in Cas9 control versus Ugcg KO NMuMG cells, as shown by GSEA. Normalized enrichment score (NES) = -1.27, P = 0.036.

GSEA of mouse EMT gene signatures in Cas9 control versus Ugcg KO NMuMG cells. NES = -2.17, P = 0.000.

Immunoblot analysis of p-SMAD2 and total SMAD2 (t-SMAD2) in NMuMG cells with or without Ugcg deficiency and treated with vehicle control or TGF-β for 1 h. Tubulin: loading control.

qRT‒PCR analysis of TGF-β target genes, including Serpine1 and Smad7, in Ugcg KO NMuMG or control NMuMG cells treated with vehicle control or TGF-β for 6 h.

Immunoblot analysis of E-cadherin and N-cadherin in Ugcg KO NMuMG or control NMuMG cells treated with vehicle control or TGF-β for 48 h. Tubulin: loading control.

Cdh1 and Cdh2 mRNA levels in Ugcg KO or control NMuMG cells after vehicle control or TGF-β treatment for 48 h.

Immunofluorescence analysis of the expression and localization of E-cadherin (red) and the formation of F-actin (green) after treatment with vehicle control or TGF-β for 2 days. Nuclei were counterstained with DAPI (blue). Images were acquired with confocal microscopy. Scale bar = 50 μm.

Data information: TGF-β was applied at a final concentration of 2.5 ng/mL. In (E, G), the data are expressed as the means ± SD values from three biological replicates (n=3). **, P < 0.01; ****, P < 0.0001 based on unpaired Student's t test.
ST3GAL5-catalysed gangliosides inhibit TGF-β-induced epithelial-mesenchymal transition via TβRI degradation (2022)
Figure 3

Figure 3. Eliglustat, a UGCG inhibitor, enhances TGF-β signaling and TGF-β-induced EMT, migration, extravasation and early metastatic outgrowth of A549 cells.

Immunoblot analysis of p-SMAD and t-SMAD2 in A549-VIM-RFP cells pretreated with eliglustat for 4 or 6 days and then treated with vehicle control or TGF-β for 1 h. GAPDH: loading control.

Quantification of the p-SMAD2 level in A549-VIM-RFP cells as shown in (A). The levels were normalized to that of GAPDH, and fold changes were then further normalized to the level of p-SMAD2 in control cells without TGF-β treatment.

Immunoblot analysis of the epithelial marker E-cadherin and mesenchymal markers N-cadherin and vimentin in A549-VIM-RFP cells treated with eliglustat for 4 or 6 days and/or with vehicle control and/or TGF-β for 2 days. Tubulin: loading control.

Effect of eliglustat (pre)treatment for 4 days on vimentin expression in A549-VIM-RFP cells in response to TGF-β and/or SB505124 (SB, 1 μM) treatment for the indicated times. The time course of RFP-tagged vimentin expression was monitored with IncuCyte. The red object intensity was normalized to the red intensity at 0 h.

A549-VIM-RFP cells were pretreated with eliglustat for 4 days and were then incubated with TGF-β or SB505124 (SB, 1 μM) for the indicated times. The real-time scratch assay results were analyzed with an IncuCyte system.

mCherry-labeled A549 cells were pretreated with eliglustat (2 μM) for 4 days and were then injected into ducts of Cuvier of zebrafish embryos. Representative images with magnified regions (outlined with dotted squares) of extravasated cells were acquired 4 days after injection by confocal microscopy. SB group zebrafish were treated with the inhibitor SB505124 (1 μM) in the egg water together with eliglustat for 4 days after injection with daily refreshment of the treatments. Scale bar = 300 μm or 150 μm.

Quantification of the number of extravasated cell clusters from 28 embryos per group. ****, P < 0.0001 based on unpaired Student's t test from two biological replicates (n=2).

Kaplan-Meier analysis of metastasis-free survival in eight mice in the group injected with untreated A549-Luc cells and eight mice injected with cells pretreated with eliglustat for one week. The log-rank test was used for statistical analysis; P = 0.06.

Analysis of the in vivo imaging system (IVIS) values from the fourth week post-injection in 8 mice in the control group and 8 mice in the eliglustat pretreatment group.

Representative whole-body bioluminescence images (BLI) at 10 weeks of mice injected with untreated A549-Luc cells or cells pretreated with eliglustat for one week.

Data information: TGF-β and eliglustat were applied at final concentrations of 2.5 ng/mL and 2 μM, respectively. All data are expressed as the mean ± SD values from three biological replicates (n=3). *, P ≤ 0.05; **, P < 0.01; ****, P < 0.0001. In (B, D, E), statistical analysis was based on two-way ANOVA.
ST3GAL5-catalysed gangliosides inhibit TGF-β-induced epithelial-mesenchymal transition via TβRI degradation (2022)
Figure 4

Figure 4. Inhibition of GSL biosynthesis decreases the TβRI level in lipid rafts and inhibits the ubiquitination and degradation of TβRI.

Lysates from NMuMG control cells (Cas9-expressing cells) and Ugcg KO cells were subjected to sucrose density gradient ultracentrifugation. The expression levels of flotillin-1, β1-integrin, EEA1, and TβRI in the sucrose gradient fractions were analyzed by immunoblotting. Fractions 3, 4, and 5 contained lipid rafts, whereas fractions 10-12 corresponded to the non-lipid raft fractions.

Quantification of TβRI percentages in lipid raft and non-lipid raft fractions from Ugcg KO NMuMG and control cells.

Isolation of lipid rafts from other cellular components in A549-VIM-RFP cells treated with the UGCG inhibitor eliglustat (2 μM) for 6 days using sucrose density gradient ultracentrifugation and measurement of flotillin-1, β1-integrin, EEA1, and TβRI levels in the sucrose gradient fractions using immunoblot analysis. Fractions 3-6 contained lipid rafts, whereas fractions 10-12 corresponded to the non-lipid raft fractions.

Quantification of TβRI percentages in lipid raft and non-lipid raft fractions from A549-VIM-RFP cells treated with eliglustat (2 μM) for 6 days.

Ubiquitination of TβRI was detected by immunoprecipitation (IP) of Myc-tagged caTβRI from HA-Ub-transfected HEK293T cells with or without eliglustat (2 μM) treatment for 6 days. All groups were treated with MG132 (5 μM) for 6 h.

Immunoblot analysis of TβRI expression levels in control and eliglustat (2 μM)-treated A549-VIM-RFP cells treated with 50 μg/mL CHX for the indicated times. Vinculin: loading control.

Quantification of TβRI expression levels with normalization to the t=0 controls.

Data information: In (B, D), the data are expressed as the mean ± SD values from two biological replicates (n=2). In (G), the data are expressed as the means ± SD values from three biological replicates (n=3). *, P ≤ 0.05; **, P < 0.01; ****, P < 0.0001 based on unpaired Student's t test.