EMBO/SourceData huggingface dataset:
Figure 5(A) Mice were treated with either gemcitabine (day 0, 3), anti-PD-L1 (day 4, 7, 10 and 13; black arrows), 12 Gy (day 4) alone or in combinations, as indicated (n=8 mice per group). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 is shown (means ± SD, n=1, One-way ANOVA, Bonferroni test) No weight loss was observed in the in vivo experiment.RT plus anti-PD-L1 (B) reverses T-cell exhaustion . Bar graphs show quantitation of T cell activation markers in tumor samples based on flow cytometry analysis, as indicated (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).RT plus anti-PD-L1 (C) increases CD8:Treg ratio. Bar graphs show ratio of CD8:Treg ratio in tumor samples based on flow cytometry analysis, as indicated (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).
Figure 6(A) Representative images of livers from the different treatment groups, as indicated. The corresponding liver weights are shown below (n=6 mice per group).(B) Flow cytometry analysis of CD45+CD8+, CD11b+Gr1+ and CD4+CD25+FOXP3+ cell populations in the livers of the mice from the different treatment groups, as indicated (n=5 per group; (means ± SD, n=1, One-way ANOVA, Bonferroni test).
Figure 1(C) Co-immunoprecipitation of Cdc4 using either unphosphorylated or Clb5-Cdk1-phosphorylated Far1(85-150) as bait. The experiment was repeated twice, a representative example is shown.(D) Time-lapse fluorescence images showing the expression and degradation of wild-type and S87A S91A mutant Far1(1-150)-GFP and nuclear-cytoplasmic shuttling of Whi5-mCherry. Nuclear export of Whi5-mCherry takes place at 0 min (Start point) and was used to synchronize the cells for quantitative analysis.(E, F) Mean±SEM fluorescence intensities of the indicated Far1(1-150)-GFP mutants over the cell cycle.(G) Time from Start to degradation of 50% of the indicated Far1-GFP sensor in single cells. The bars show median ±95% confidence intervals. The numbers above the plot show median values. **** indicates p-value <0.0001, *** <0.001 and ns >0.05 for pair-wise comparisons with wild-type using Mann Whitney U test.(H) In vitro kinase assay showing the effect of N130A, L131A, L135A and L136A mutations on Far1(1-150)-GFP phosphorylation by Clb5-Cdk1. WT/mut shows the decrease in phosphorylation rate of the mutant compared to wild-type. The experiment was repeated twice, a representative example is shown.
Figure 2(A) Autoradiograph of a cyclin specificity analysis of the indicated substrates with wild-type or docking site mutant cyclin-Cdk1 complexes. Far1(1-150 mut) is L131A L135A L136A mutant, Sic1ΔC is the non-inhibitory mutant of Sic1 (deletion of 216-284). The experiment was repeated twice, a representative example is shown.(B) Mean±SEM fluorescence intensities of wild-type and NLxxxL motif mutant Far1(1-150)-GFP in either wild-type or clb5(hpm) clb6(hpm) strain over the cell cycle.
Figure 3(C) Plot showing the mean±SEM fluorescence intensities of Far1(1-150)-GFP constructs with the indicated docking site. In rxl mutant, the NLxxxL motif is replaced with a mutated non-functional RxL motif (VKATAF).(D) Kinetic characterization of Far1(1-150 WT)-GFP and Far1(1-150 RxL)-GFP phosphorylation by Clb5-Cdk1. The data is from two experimental replicates.(E) Mean±SEM fluorescence intensities of wild-type or RxL-dependent Far1(1-150)-GFP in wild-type or the indicated cyclin hpm mutant strains.(F) Pheromone sensitivity halo assay with PGAL1-3HA-CLB5 sic1Δ strains carrying either wild-type FAR1, far1Δ, far1(L135A) or far1(RxL). Different concentrations of α-factor were pipetted on the paper discs. On glucose plates, α-factor triggers cell cycle arrest. CLB5 overexpression from PGAL1 in sic1Δ strain causes lethality presumably by inhibition of replication licensing, which is rescued by the presence of α-factor, which leads to Far1-depedent inhibition of excess Clb5-Cdk1 activity. The experiment was repeated twice, a representative example is shown.
Figure 4(B) The degradation of Far1(1-150)-GFP with either wild-type or all serine-based phosphorylation sites and the indicated docking motifs was measured in time-lapse microscopy. Plot shows mean±SEM of GFP fluorescence intensities from Start.(C) Phos-tag SDS-PAGE autoradiographs showing the Cks1-dependency of Clb5-Cdk1-mediated multisite phosphorylation of Far1(1-150) with either NLxxxL (WT) motif or RxL motif. The experiment was performed twice, a representative example is shown.(D) Plot showing the kinetics of Clb5-Cdk1-mediated phosphorylation of Far1(85-150) with either wild-type (NLxxxL) or RxL docking motif. The data is from two replicates.(F) Mean±SEM GFP fluorescence intensities of the indicated Far1(1-85) constructs, synchronized at the time of Start.(G) Plot showing the timing of 50% degradation of the indicated Far1(85-150) constructs in single cells. The numbers above the plot show median values. The bars show median ±95% CI. **** and ns indicate p-values <0.0001 and >0.05 by Mann-Whitney test for pair-wise comparisons with WT.
Figure 5(C) Plot showing the mean±SEM fluorescence intensities of the Sic1-based threshold sensors shown in 'A' containing the indicated cyclin docking motif.(D) The time from Start to the fluorescence levels of the threshold sensors with indicated docking motifs decreasing to 50% of peak level was measured in single cells. The black lines show median±95% CI. The numbers above the plot show median degradation timings of the indicated sensors.(E) In vitro phosphorylation kinetics of Sic1(T33+16T45 rxl llpp ΔC) with the indicated cyclin docking motif by Clb5-Cdk1. The data is from two replicate experiments.(F) Analysis of multisite phosphorylation of Sic1(T33+16T45 rxl llpp ΔC) with different docking motifs using Phos-tag SDS-PAGE autoradiography. The experiment was performed twice, a representative example is shown.(G) The fraction of maximally phosphorylated form from total phosphorylation signal was quantified from Phos-tag gels as shown in panel 'F'.
Figure 6(B) Autoradiograph showing the cyclin specificity and docking dependency of Lif1 and Slx4 phosphorylation. The experiments were repeated twice, representative examples are shown.(C) NLxxxL is necessary for efficient phosphorylation of Lif1 and Slx4 by Clb5-Cdk1, as shown by the in vitro kinase assay. The experiments were repeated twice, representative examples are shown.(D) Autoradiograph of Clb5-Cdk1-mediated phosphorylation of indicated Fin1 mutants. The experiments were repeated twice, representative examples are shown.(G) The effect of different docking motifs on the degradation of Far1(1-150 SP)-GFP, where all TP sites are mutated to SP. Plot shows mean±SEM of fluorescence intensities in the cell cycle.(H) The time from Start to the degradation of 50% of Far1(1-150 SP)-GFP with the indicated docking motif was measured in single cells. The numbers above the plot show median values for the sensors, the black lines show median±95% CI. **** and ns denote p-value <0.0001 and >0.05, respectively, for pair-wise comparisons with SP WT using Mann-Whitney U test.
Figure 7(A) Time-lapse microscopy was used to measure cyclin-Citrine levels during mitotic exit in unperturbed cell cycles. The cells were synchronized by the nuclear import of 50% of Whi5-mCherry at mitotic exit. The plot shows mean ±SEM fluorescence intensities averaged over a population of cells. The vertical line at -14 minutes marks the estimated time of anaphase entry.(B) Cyclin-Citrine degradation relative to the nuclear import of 50% Whi5-mCherry was followed in single cells. Plot shows the time from nuclear import of 50% Whi5-mCherry to the degradation of 50% of the indicated cyclin in individual cells. The error bars show 95% CI of the mean.(E) The effect of cyclin specificity on the phosphorylation of Fin1, Cnn1 and Spc110 was studied in a kinase assay. The plots show the phosphorylation by Clb5-, Clb3- and Clb2-Cdk1, the error bars show standard deviation (n=2).(F) Images from a time-lapse microscopy experiment of an exemplar cell expressing Spc42-mCherry for detection of SPBs and EGFP-Fin1. Cells were imaged every 2 minutes, the white arrow indicates onset of anaphase.(G) The accumulation of EGFP-Fin1 to SPBs in anaphase was measured in experiments described in 'F'. The plot shows mean EGFP fluorescence intensities at SPBs around the time of anaphase onset. The error bars are ±SEM.
Figure 1Expression 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.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.
Figure 2Quantification 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).Transversal section of a recombined VeCadCreER:Confetti AGM from an E10.5 embryo and immunostained with CD31 (C) (white in image, black in label) Multistack reconstruction of confocal images. Scale bars 30μmTransversal section of a recombined VeCadCreER:Confetti AGM from an E10.5 embryo and immunostained with Kit (D) (white in image, black in label). Multistack reconstruction of confocal images. Scale bars 20 μmAnalyses 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).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.
Figure 3Detail 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.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).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
Figure 4(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.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).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)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.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).
Figure 5Experimental 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.Experimental design and sorting gate for Kit+CD45- cells for single cell RNAseq (n=483 cells).Violin plots of Notch family elements expression in IgG and αDll4 treated cells (adj p val: *<0.1 and *** p<0.00001).Percentage of cells in each cell cycle phase extracted from single cell RNA-seq analysis. Bars represent the average±SE (n=483).
Figure 6Bar 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).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.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).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. Quantification of recombined cells in the aortic endothelium using confocal images. Mean±SE (n=3).
Figure 7Snapshots 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.
Figure 8Colony-forming assays using E10.5 AGMs derived from embryos treated with IgG or αDll4.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.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.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.
Figure 2B. DIC microscopic image of WTSOD12SH diluted to 100 µM in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated 37 °C, 180 RPM for 30 min shows no droplet formation; scale bar: 100 µm; inset shows Alexa Fluor 488 maleimide labelled WTSOD12SH;C. Solution turbidity plot (absorbance at 600 nm) shows that WTSOD12SH (at concentrations ranging from 25 to 200 µM does not undergo LLPS in presence or absence of heparin, LLPS inducer; data is represented as mean ± SD (from 3 biological replicates).D. DIC microscopic image of ApoSOD12SH under condensate inducing conditions (37 C, 180 RPM) at a concentration of 100 µM incubated 100 mM NaCl; scale bar:100 µm (inset on bottom left shows Alexa Fluor 488 maleimide labeled protein condensates;E. Fluorescent microscopic images of Alexa Fluor 488 maleimide labeled ApoSOD12SH condensates diluted in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated (180 RPM, 37 C) in absence (left) and presence (right) of heparin at 30 min time-pointF. Solution turbidity measurements with ApoSOD12SH (absorbance at 600 nm) shows that while in the absence of heparin the turbidity increases with time, the presence of heparin results in faster LLPS;G. Comparison of ApoSOD12SH droplet numbers per microscopic field of view (view area 0.001963 mm2) calculated from 5 different images of droplets incubated with and without heparin at 2 h timepoint.H. Liquid nature of ApoSOD12SH showing droplet fusion and surface wettingI. Amplitude normalized FCS curves of ApoSOD12SH (in blue) of dilute phase (DP) and condensed phase (CP). Intense color indicates CP and light variant indicates DP. The increase in τD (as shown by the arrow) translates to a decrease in DJ. Scheme depicting that presence of Zn and not Cu in pre-incubation mixture inhibits LLPS. K. DIC microscopic images of ApoSOD12SH in presence of Zn (top) showing no droplet formation and Cu (below) showing the presence of dropletsL. Solution turbidity plot (absorbance at 600 nm) of ApoSOD12SH subjected to increasing concentrations of Cu and Zn; turbidity decreases in a dose dependent manner for Zn while no significant change is observed in presence of Cu;
Figure 3A. DIC microscopic images of I113T SOD12SH (top) and G85R SOD12SH (bottom) droplets incubated in the absence and presence of Zn and Cu respectively (scale bar: 100 µm); insets on bottom left show Alexa Fluor 488 maleimide labeled protein condensates for I113T SOD12SH and G85R SOD12SH respectively; scale bar: 5 µm. Droplet dissolution was observed for I113T SOD12SH on addition of Zn while droplets persisted on addition of Cu. Red arrowhead indicates fusion of liquid droplets.B. Amplitude normalized FCS curves of I113T SOD12SH (in green) and G85R SOD12SH (in grey) of dilute phase (DP) and condensed phase (CP) showing an increase in diffusion time from DP to CP. The intense color indicates CP and light variant indicates DP.C. On metal cofactor removal, ApoG37R SOD12SH formed condensates which ceased to exist when protein was incubated with Zn, while condensates persisted on Cu addition (left to right);
Figure 4B. Plot of the loge of the hydrodynamic radius (rH) versus the log e of the number of residues in the polypeptide chain. The line fitted to these data for the native folded proteins (yellow dashed) has a slope of 0.29 ± 0.02 and a y-axis intercept of 1.56 ± 0.1, while the other fitted to the chemically denatured protein (grey dashed) data has a slope of 0.57 ± 0.02 and a y-axis intercept of 0.79 ± 0.07. Literature data have been used for folded and chemically denatured proteins while we employed FCS to calculate the rH of all SOD12SH variants with (solid shapes) and without Zn (hollow shapes). The hollow grey circle of G85R SOD12SH is directly under hollow green I113T SOD12SH in the log-log plot.C. Variation of diffusion coefficients of de-metalated protein variants determined from FCS measurement with increasing Zn concentration (inset shows a comparison of binding constants between mutants and Zn; ApoSOD12SH and ApoG37R SOD12SH has a higher binding affinity to Zn than ApoI113T SOD12SH and ApoG85R SOD12SH).D. Disordered/ extended conformation content decreases in ApoSOD12SH and ApoI113T SOD12SH with the addition of Zn calculated from FTIR spectra;
Figure 5C. Salt-bridge analysis of ApoG85R SOD12SH (dist. cutoff <0.5 nm = salt-bridge).
Figure 6C. Upper panel shows fluorescence images of the maturation of Alexa Fluor 488 labeled ApoSOD12SH droplets with time, samples were incubated at 37 C up to ~25 hours. Images are taken at indicated time point: 0* hour (the time after incubation required for droplet formation), 12 hours and >24 hours; images below show magnified scale bar: 5 µm. The middle panel shows magnified view of the marked region from corresponding images on the upper panel. Red arrowhead indicates matured (solid) portion inside the droplet. Lower panel shows coexistence of liquid and solid phase inside droplet; scale bar: 10 µm. Intensity plot (bottom right) shows the distribution of high intensity region within matured droplet.D. Diffusion coefficient obtained from point FCS measurement at different regions of the droplet during maturation course; 0* (x-axis label) indicates the time after incubation required for droplet formation.E. ThT fluorescence assay plot showing aggregation kinetics of all SOD12SH variants. ApoSOD12SH, I113T SOD12SH and G85R SOD12SH show high ThT fluorescence.F. AFM micrographs of ApoSOD12SH, I113T SOD12SH and G85R SOD12SH show fibrillar aggregates after prolonged incubation at 37 ℃, 180 RPM in absence of Zn (left panel); AFM micrographs of ApoSOD12SH, I113T SOD12SH and G85R SOD12SH incubated with Zn (at 1:5 protein: Zn molar ratio) show presence of oligomers for ApoSOD12SH, I113T SOD12SH and short fibrils for G85R SOD12SH (right panel);G. Cell proliferation of SHSY5Y human neuroblastoma cells assessed by MTT assay post 12 h treatment with 5 µM ApoSOD12SH, I113T SOD12SH and G85R SOD12SH condensates and aggregates (fibrils). Cell culture medium (DMEM) was used as negative control and untreated SHSY5Y cells were used as positive control.H. Dot plots showing flow cytometric analysis of annexin V and propidium iodide (PI) staining of apoptotic SHSY5Y neuroblastoma cells following a 12 h treatment with 5 µM ApoSOD12SH condensates, I113T SOD12SH condensates, G85R SOD12SH condensates, ApoSOD12SH fibrils, I113T SOD12SH fibrils and G85R SOD12SH fibrils respectively (see Methods for details). High cytotoxicity was observed for SOD12SH condensates.
Figure 1Coomassie Blue staining of recombinant human DDX5 purified in bacteria. M denotes the molecular mass markers in kDa.R-loop unwinding assay in presence of increasing DDX5. The top panel shows a typical image obtained after the assay. The bar graph (bottom) shows the quantification. The average is expressed as percentage unwinding, and standard error of the mean (S.E.M.), n=4.S9.6 signal in the nucleoplasm of U2OS cells. An average from 3 independent experiments performed in triplicate. In total, ~90 images for each condition (10 images/slide and 9 slides for each condition) were taken and two cells per image were quantified. Statistical significance was assessed using one-way ANOVA t-test. *: p < 0.05; ****: p < 0.0001.U2OS cells transfected with siCTL, or siDDX5 were subjected to DRIP-qPCR analysis with anti-IgG, and anti-S9.6 antibodies with or without RNaseH treatment. The gene location and genomic qPCR amplification region are shown at the top of each panel. B, E, H, S and X denote the location of the BsrGI, EcoRI, HindIII, SspI, and XbaI. The identified R-loop peaks were extracted from the R-loop database (R-loop DB) for each region. The bar graphs are the average and S.E.M. from 3 independent experiments. Statistical significance was assessed using t-test. *: p < 0.05.
Figure 2U2OS cells were lysed and the lysates were incubated with anti-PRMT5 antibody or immunoglobulin G (IgG) as control followed by protein A Sepharose bead addition. The immunoprecipitated (IP) proteins or whole cell extract (WCE) were separated by SDS-PAGE and Western blotted (WB) with the indicated antibodies. Same as panel (A) except anti-DDX5 antibody was used instead of anti-PRMT5 antibody for immunoprecipitation.The mass spectrum of R502 monomethylation from Flag-DDX5 expressed in U2OS.HEK293 cells transfected with Flag-DDX5 were lysed and immunoprecipitated with anti-Flag antibodies. The bound proteins were along with WCE from untransfected (-) and Flag-DDX5 (+) transfected cells were Western blotted with anti-FLAG, anti-methylarginine antibodies (MeR).The knockdown using siRNAs of the indicated gene in U2OS cells was confirmed by Western blottingThe knockdown using siRNAs of the indicated gene in U2OS cells were analyzed by immunofluorescence with S9.6 and anti-nucleolin antibodies. A typical image is shown.