EMBO/sd-character-level-ner huggingface dataset:
Figure 10Measured Log2(H/M) values at four time points as a function of their half-lives estimated here and elsewhere (Cohen et al, 2013). All log2(H/M) values shown here are averages of values obtained in 5 experiments (3 using lactacystin, 2 using epoxomicin). Proteins for which statistically significant differences at t=24 hours were observed (P<0.05, two-tailed Welch's t-test) are shown in red. Expected log2(H/M) values based on equation 4 are plotted as light blue lines. To minimize the masking of potential dependencies by the slight imperfections in the H/M normalization process (which introduces small offsets along the Y scale), average population log2(H/M) values of each time point (Fig. 6E) were subtracted from all measured Log2(H/M) values. Data for 174 synaptic proteins for which half-life estimates were available.
Figure 8 A-D Sensitivity to acute MMS treatment. Log-phase cultures of rad18Δ (TB058), rev3Δ (TB060), scc2-4 (TB073), scc1-73 (TB078), scc2-4 rad18Δ (TB082), scc2-4 rev3Δ (TB083), scc1-73 rad18Δ (TB086) and scc1-73 rev3 (TB087) were treated with indicated concentrations of MMS for 1 hour. Next, cells were washed and plated on solid YPD. Error bars represent mean value ± standard deviations of mean (n=3)  E-H Levels of MMS-induced Rfa1-YFP and Rad52-YFP foci. Logarithmically growing cultures of rad18Δ (TB052 or TB058), rev3Δ (TB054 or TB060), scc2-4 (TB071 or TB073), scc1-73 (TB076 or TB078), scc2-4 rad18Δ (TB080 or TB082), scc2-4 rev3Δ (TB081 or TB083), scc1-73 rad18Δ (TB084 or TB086) and scc1-73 rev3 (TB085 or TB087) were treated with MMS for 1 hour at 25°C. Next, cells were washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=3)
Figure 5By tracking the patients' heterogeneous drug response caused by different genetics and environments, adaptive chronotherapeutics adjusts the dosing time: if the current dosing regimen leads to a weaker or stronger drug effect than the desired one, the dosing time is delayed or advanced by 1 h, respectively B, C By adjusting the initial dosing time (ZT3) according to the adaptive chronotherapeutics, the precise dosing time can be obtained for each ASPD model (colored number; B), with which a single daily 10 mpk dosing leads to the desired ~4 h phase delay in LD 16:8 (gray range; C). D, E When day length decreases to 8 h (LD 8:16), the precise dosing times are considerably advanced.
Figure 4(A) Stretched DNA was incubated in the HSS in the presence or absence of geminin. After washing the HSS, endogenous Smc3 (green) and Mcm2 (magenta) were detected by immunofluorescence staining. DNA was counterstained with SYTOX (gray). Scale bar, 2 µm.(B) Numbers of Smc3 and Mcm2 signals were counted on 10 μm DNA shown in (A). (22 DNAs per condition, n = 3, mean ± s.e.m. *p < 0.0001, two-tailed Mann-Whitney test). (C) Chromatin formed in the HSS in the presence of geminin, and cohesinHalo488 (green) was loaded onto the chromatin with Scc2-Scc4. Chromatin was detected by immunostaining of histone H3 (magenta). DNA was counterstained with SYTOX (gray). The distributions of cohesin particles and histone H3 are shown as line-scan plots (bottom). Scale bar, 5 μm.(D) Cohesin particles observed in (C) were categorized as indicated (> 37 particles per condition. n = 6, mean ± s.e.m.).(E) Cohesin particle numbers for each condition. Red bars denote the median, lower, and upper quartile values (n ≥ 12, *p < 0.0004, unpaired t-test).(F) Colocalization of Mcm2 with Smc3 was quantified (50 Mcm2 particles per condition, n = 3, mean ± s.e.m.)
Fig.Figure 3A) Gene set enrichment analysis of the transcriptome data from uninfected vs. 48 h SARS-CoV-2 infected organoids was performed and the GO terms with the most significant positive correlation are displayed. B) Gene set enrichment analysis of the transcriptome data comparing uninfected vs. 48 h SARS-CoV-2 infected organoids and the "Hallmark interferon gamma response" gene set. p-values were calculated using the fgsea package from https://www.bioconductor.org with 5000 permutations. C) Analysis of discordance and concordance of transcriptomic responses (DISCO) between untreated vs. SARS-CoV-2 infected and untreated vs IFN-γ treated organoids. The 50 most regulated genes are highlighted; grey: discordant and black: concordant. D) Relative expression of selected enterocyte-specific genes upon SARS-CoV-2 infection, normalized to GAPDH. Differences in gene expression were assessed using the linear model 'lmFit' and 'makeContrasts' in limma. E) Immunofluorescence for KRT20 (green) in human colon organoids cultured in basic medium and either non-infected (left) or 48 h after SARS-CoV-2 infection (right), indicating a strong increase in KRT20 expression after viral infection. Scale bar: 50 µm. Quantification of fluorescence intensity below (n=8). Data are presented as mean +/- SD ,****: p≤0.0001, as determined by Student's t-test F) qPCR data displaying the increase of INF-γ target genes (CXCL11, IFIT3) (upper) and enterocyte marker genes (KRT20, ACE2) (lower) after SARS-CoV-2 infection, validating the results from the microarray analysis (n=3). Data are presented as mean +/- SD ,****: p≤0.0001, as determined by Student's t-test
Figure 6A. Cartoon representation of reaction conditions used in (B-D), which simultaneously enable de novo chain assembly as well as the formation of K48/K63-branched chains. B. In vitro ubiquitination reactions with full‑length Ubc1 or Ubc1-ΔUBA were performed. Samples were taken at indicated time points and analyzed by SDS-PAGE and fluorescence scan. Free chain synthesis from Alexa488-labeled monoubiquitin was observed over time. Fluorescence of all reaction products was quantified and normalized to number of fluorophores per product ("de novo"). Double cross (‡) indicates ubiquitinated E2 enzyme. C. Reactions as in (B) were performed in presence of C‑terminally capped and Alexa647-labeled K63‑linked Ub3. Monoubiquitin and K63-Ub3 were used in a 3:1 molar ratio. Analysis as in (B). Modification of K63-Ub3 was identified through correlation of fluorescence channels and their fluorescence quantified in the 488 channel ("branched"). D. Alexa488-labeled monoubiquitin and C-terminally capped K63-Ub4 were used in a 4:1 molar ratio for in vitro ubiquitination reactions with full‑length Ubc1 or Ubc1-ΔUBA. Where indicated, Alexa488-labeled monoubiquitin was methylated prior to the reaction (met-Ub) to block de novo chain assembly. Reactions were incubated overnight, quenched and half the reaction mix from each condition was treated with the K63 specific deubiquitinating enzyme AMSH (lanes 4-6). Samples were analyzed by SDS-PAGE and fluorescence scan.
Rolling behavior and rheotaxis of mouse sperm.Figure 5Left: Bright-field image series of a freely swimming wild-type sperm cell at t = 0, 151, and 303 ms. Scale bar represents 10 µm. Right: Rotation frequency (mean ± SD) of 24 freely swimming wild-type sperm. Left: Bright-field image series of a freely swimming Catsper1-/- sperm cell at t = 0, 151, and 294 ms. Scale bar represents 10 µm. Right: Rotation frequency (mean ± SD) of 24 freely swimming Catsper1-/- sperm. Representative trajectories of wild-type sperm in the absence of a fluid flow. The starting point of each trajectory was centered to the origin of a coordinate system, represented by the intersection of the dotted lines in the center of the circle. Each color represents one trajectory. Spider-web plot of the mean (± SD) angular frequencies (binning: 315°-45°, 45°-135°, 135°-225°, and 225°-315°; 127 sperm; 3 experiments) in the absence of a fluid flow. Representative trajectories of wild-type sperm in the presence of a fluid flow. Spider-web plot of the mean (± SD) angular frequencies (175 sperm; 3 experiments) of wild-type sperm in the presence of a fluid flow. The red arrow indicates the flow direction. Representative trajectories of Catsper1-/- sperm in the absence of a fluid flow. The starting point of each trajectory was centered to the origin of a coordinate system, represented by the intersection of the dotted lines in the center of the circle. Trajectories are magnified by a factor of 2.05 with respect to the plots C and E to compensate for the reduced swimming speed of the Catsper1-/- sperm. Spider-web plot of the mean (± SD) angular frequencies of Catsper1-/- sperm (297 sperm; 4 experiments) in the absence of a fluid flow. Representative trajectories of Catsper1-/- sperm in the presence of a fluid flow; Trajectories are magnified by a factor of 2.05 with respect to the plots C and E to compensate for the reduced swimming speed of the Catsper1-/- sperm, two trajectories were truncated (indicated by two parallel lines). Spider-web plot of the mean (± SD) angular frequencies (261 sperm; 4 experiments) of Catsper1-/- sperm in the presence of a fluid flow. The red arrow indicates the flow direction.
TCHP contributes to autophagosome maturation A, The postnuclear fraction (PNS) from TCHP knock-down and control HUVECs in the presence or absence of BafA1 was separated into (low-speed pellet) LSP and (high-speed pellet) HSP fractions and then analysed by immunoblots using anti p62, LC3 and GABARAP antibodies.Figure 4The postnuclear fraction (PNS) from TCHP knock-down and control HUVECs in the presence or absence of BafA1 was separated into (low-speed pellet) LSP and (high-speed pellet) HSP fractions and then analysed by immunoblots using anti p62, LC3 and GABARAP antibodies. The sub-fractions were treated with proteinase K (Prot. K) with or without Triton X- 100 (TX-100). Quantification of the Western blot: the p62, LC3 and GABARAP levels relative to respective GAPDH were quantified using densitometry analysis and normalized to the value of non-treated samples. Representative TEM images of autophagosomes in control and TCHP knock-down HUVECs in the complete medium after 3h incubation with BafA1 (Scale bars, 600nm): i, quantification of mean autophagic vesicles (AVs) and autophagosome (APs) area and ii, distribution of the cross-section areas of the analysed vesicles expressed in percentage. (n=7 cells; >100 vesicles per sample; AVs: unpaired t-test; **p<0.0001 vs control; APs: unpaired t-test **p<0.0001 vs control). Left panels: Representative single-channel and merged images of HUVECs expressing GFP-LC3 and immunostained for STX17. Scale bars, 25 µm and 2 µm in the inset. Right panels: Quantification of LC3/STX17 positive cells in the presence or absence of BafA1 (one-way ANOVA; **p=0.0060 vs control; #p=0.0136 vs control BafA1) and quantification LC3/STX17 positive puncta per cells (n=50 cells; one-way ANOVA; **p<0.0001 vs shTCHP BafA1).
R1-6 photoreceptors are required for lamina glia maintenance.Fig 3(A) Rh1>lacZ+H2B-RFP exhibited nuclearRFP expression in the R1-6 photoreceptors (red). (B) Rh1>lacZ+Shits1 maintained at 29°C for 14 days resulted in lamina vacuolization. (C) Pan-Rh7>H2B-RFP exhibited expression in R7 (arrow) and R8 (arrowhead) (red). (D) Pan-Rh7>Shits1 incubated at 29°C for 14 days did not exhibit lamina degeneration. (E) Lamina monopolar (L) neurons (red) were selectively labeled by the Ln-GAL4 driven H2B-RFP with repo-GAL80. (F) Ln>Shits1, repoGAL80 shifted to 29°C for 14 days did not exhibit lamina degeneration. (G) R1-6 photoreceptors were killed in Rh1ts>lacZ+Hid shifted to 29°C for 14 days. Rh1ts indicates tubGAL80ts; Rh1-GAL4. Degeneration was induced in the lamina in addition to the retina. (H) rdgC306/+ mutant exposed to constant light for 14 days exhibited degeneration in the retina, as well as the lamina. DAPI: nuclei (white in A-H). (I, K) Rh1>lacZ+Shits1 at 29°C for 4 days exhibited vacuoles in the electron dense cytoplasm of epithelialglia or near the glianucleus. Arrow: vacuole with internal debris. Arrowhead: large vacuole. (J) Lamina cartridge of Rh1>lacZ+H2B-RFP. N indicates glialnuclei. (L) The percentage of the vacuole area in the lamina at 29°C for 14 days was examined. All P-values were calculated using one-way ANOVA with Tukey's post-test. Scale bar: 20 μm.
Figure 2Mabs-laden wet-spun yarns showing progressive myogenic differentiation over culturing time. As can be noted, Mabs morphology evolves from a round shape (single cells at early time points) into a more elongated one (generally within the first 3-5 days of culture), eventually forming long-range myotubes. For the sake of clarity, samples used for bright field investigation were prepared out of few layers of fibers to favor the recognition of cell organization.
Figure 4A. Experimental design for pIpC treatment and analysis of PKCδ fl/fl/Mx-1Cre- (PKCδ fl/fl) and PKCδ fl/fl/Mx-1Cre+ (PKCδ Δ/Δ, cKO) mice. B,C Representative FACS plots of PKCδ fl/fl and cKO BM 4-8 weeks (B), and 20-24-weeks (C) after pIpC treatment. Numbers in plot indicate the absolute numbers of the indicated subset within the gated population (n=8-9 mice per genotype and time point). Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis. D. Schematic of competitive BM transplants. Total BM cells from either PKCδ fl/fl/Mx-1Cre- (control) or PKCδ fl/fl/Mx-1Cre+ mice were injected together with recipient type (CD45.1) BM cells (1:1) into lethally irradiated CD45.1 mice prior to pIpC treatment. Two months after transplant, both groups of mice received five doses of pIpC. Chimeras were analyzed at 16-weeks after the last pIpC injection. (E) FACS plots show contribution of donor-derived (CD45.2+) HSPCs in recipient BM (E) (total of n=7-8 mice per genotype). Calculated absolute numbers of donor-derived HSPC subsets in transplanted mice (F). (total of n=7-8 mice per genotype). Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis. G. Average percentage BrdU+ HPSCs (3 days after BrdU injection into mice reconstituted 24 weeks previously with control or cKO marrow and injected 16 weeks previously with pIpC among donor-derived LSK cells in the control and cKO chimeras BM. Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis. H. Percentages of apoptotic cells (AnnexinV+/7-AAD-) in donor-derived LSK cells in control and cKO chimera mouse BM at 24 weeks after transplant and 16 weeks after pIpC injection. Data are compiled from two independent experiments (total of n=7-8 mice per genotype). Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis.
Figure 7A, Immunoblot analysis of protein lysates from ARPE-19 cells over-expressing FLAG-tagged C. elegans HLH-30 wild-type or mutated HLH-30(C284A) mutant. Samples were run under non-reducing conditions. B, Immunoblot analysis of protein lysates from C. elegans knock-in strains expressing HLH-30::3xFLAG::eGFP and HLH-30(C284A)::3xFLAG::eGFP subjected to 4 h of starvation. Samples were run under non-reducing conditions. C, Representative examples of HLH-30::3xFLAG::eGFP subcellular localization: Cytoplasmic, diffuse GFP distribution along the whole body of the worm; Weak nuclear, some cells in the posterior part of the worm display nuclear GFP labeling but no nuclear labeling is found in the head cells; Strong nuclear, distinct nuclear GFP labeling in cells along the worm body, including neurons in the head area. Arrowheads denote nuclear localization of HLH-30::3xFLAG::eGFP. Scale bar 200 µm and 50 µm. D, Quantification of the subcellular distribution of HLH-30::3xFLAG::eGFP and HLH-30(C284A)::3xFLAG::eGFP upon treatment with different stimuli. Data are from three independent experiments with at least 50 animals per experiment.
Transfection of pathogen-derived ligands bypasses GBP1 in inducing macrophage death (A) Cell death measured using Annexin V (AnnV)-Glo assay of THP-1 ∆CASP1 cells transfected with non-targeting control (CTRL) or GBP1 siRNA and treated as indicated.Figure 6Cell death measured using Annexin V (AnnV)-Glo assay of THP-1 ∆CASP1 cells transfected with non-targeting control (CTRL) or GBP1 siRNA and treated as indicated. Cells were primed with IFNγ, transfected with soluble Tg antigen (STAg) or both, or left untreated (UT). Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. AnnV-Glo assay from naïve or IFNγ-primed THP-1 ∆CASP-1 cells transfected with soluble Tg antigen (STAg) that was untreated (UT) or treated as indicated. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. Immunoblots from IFNγ-primed THP-1∆CASP-1 or THP-1 ∆ASC cells transfected with non-targeting control (CTRL), GBP1, AIM2, CASP-1 or CASP-8 siRNA and then untreated (-) or transfected with STAg for 12 h. Images are representative of n = 2 independent experiments. AnnV-Glo assay from naïve or IFNγ-primed THP-1 ∆CASP-1 cells transfected with non-targeting control (CTRL), GBP1, AIM2, CASP-1 or CASP-8 siRNA and then untreated (UT) or transfected with STAg as indicated for 18 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. Schematic on right shows an overview of pathways leading to caspase-8 activation. Propidium iodide (PI) uptake assay from MDMs transfected with non-targeting control (CTRL) or GBP1 siRNA and then left untreated (UT), treated with LPS or transfection reagent (Lipofectamine) only or transfected with LPS for 4 h. Area under the curve (AUC) from real-time assays plotted as mean ± SEM from n = 4 independent experiments. Schematic on right shows an overview of pathways leading to caspase-4 activation.
Experimental setup using pSILAC and N-terminal COFRADIC to assess N-terminal proteoform stability.Figure 3A. Jurkat cells were pre-labeled with light or medium L-Arg isotopes. A label swap was performed of cells growing in medium Arg, whereas light cells were cultured in the same medium. Cells were harvested at different time points and equal proteome amounts were mixed, followed by N-terminal COFRADIC fractionation and LC-MS/MS analysis. B. Ratios between three dynamic forms of N-terminal peptides reflect protein synthesis and degradation rates, allowing the calculation of 50% turnover times. C. The distribution of turnover times (N=1,894) in our experiment was not unimodal, with a sizable number of quickly turned over proteins, with the median protein turnover rate being 21.6 h. The doubling time of Jurkat cells (24 h) is indicated in red.
Human LGP2 is essential for the induction of a type I IFN response upon depletion of ADAR1.Figure 1A) THP-1 monocytes were genetically engineered to knockout RIG-I, MDA5, or LGP2 using CRISPR/Cas9. Cells were differentiated towards macrophage-like cells using PMA and treated with recombinant type I IFN to upregulate RLR expression. Correct gene editing and intact type I IFN responsiveness was validated by SDS-PAGE and immunoblotting using the indicated antibodies (n=3). *, non-specific band. B) Cells generated in (A) were differentiated using PMA and transfected with a control siRNA (siCtrl) or an ADAR1-targeting siRNA (siADAR1). The type I IFN response was monitored 56h post-transfection by RT-qPCR analysis to determine IFN-β and IFIT1 transcript expression, normalized to a housekeeping gene (ACTB). ADAR1 knockdown efficiency was monitored by ADAR1 p110 expression, normalized to ACTB and displayed relative to siCtrl. Data are means ± s.d. from a representative of three biological replicate experiments. C) Primary human monocyte-derived macrophages were transfected with the indicated siRNAs. Cells were harvested 96h post-transfection and RT-qPCR analysis was used to monitor the type I IFN response (IFN-β, IFIT1, and ISG15 transcripts) and knockdown efficiency of ADAR1 and LGP2 (DHX58). All transcripts were normalized to ACTB. Data from two independent donors (denoted with distinct symbols) are shown with mean ± s.d.. D) HEK293 cells were genetically engineered to knockout RIG-I, MDA5, or both, and subsequently subjected to retroviral transduction to stably express FLAG-LGP2 or an empty vector (EV). Correct gene editing and intact type I IFN responsiveness was validated by SDS-PAGE and immunoblotting using the indicated antibodies (n=2). *, non-specific band. E) Cells generated in (D) were transfected with siCtrl or siADAR1. The type I IFN response and ADAR1 knockdown efficiency was monitored 78h post-transfection as in (B). Data are means ± s.d. from a representative of four biological replicate experiments. F) Cells generated in (D) were transfected with siCtrl (siC) or siADAR1 (siA). Protein lysates were prepared 78h post-transfection, followed by SDS-PAGE and immunoblotting using the indicated antibodies (n=2). G) HEK293 WT cells and FLAG-LGP2-expressing HEK293 cells were transfected with siCtrl or siADAR1 and subsequently plated on coverslips for immunofluorescence. Cells were fixed, permeabilized and stained 72h post-transfection with anti-FLAG (red) and anti-IRF3 (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar is 50 μm. Total nuclei (>450 nuclei per experimental condition) and IRF3-positive nuclei were counted using semi-automated software analysis and plotted as percentage IRF3-positive nuclei of total nuclei per field of view (a representative of three biological replicate experiments is quantified). The boxplot indicates the interquartile range as a box, the median as a central line, and the whiskers extend from the minimum to the maximum value. Statistical analyses were performed using unpaired two-tailed Mann-Whitney U tests. ns, not significant; ****, p<0.0001.
Figure 3(A) Healthy mice or BLM-induced mice were tail vein injected on day 10 with 10 nmol OTL38 in the absence or presence of 200-fold excess FA-glucosamine to block all folate receptors. After 2h, mice were euthanized, resected and imaged for fluorescence intensity (n=5). (B) Alternatively, lungs from mice injected with 100 nmol OTL38 in the present or absence of 200-fold excess FA-glucosamine were collagenase digested and stained with 7AAD plus antibodies to CD11b and F4/80 prior to FACS analysis (n=3). Representative plots showing the gating strategy yielding live macrophages (7AAD- CD11b+ F4/80+) and OTL38 positive macrophages are shown. (C) Percentages of live macrophages in BLM-induced mice (n=3). (D) Percentages of lung macrophages that accumulated OTL38 in vivo (n=3).
Figure 2Stress-induced p53 binding at human subtelomeres. (A) Western blot of HCT116 p53-/- and p53+/+ cells treated with DMSO (D) or 50 µM etoposide (E) for 24 hrs. Cell lysates were assayed for total p53, phospho-Ser15 p53 (p-p53), γH2AX, or GAPDH. (B) ChIP-qPCR was assayed in HCT116 p53-/- or p53 +/+ cells treated with DMSO or etoposide as in panel A, and assayed by ChIP with antibodies to p53 (Ab6) or control IgG, followed by qPCR for p53 binding site in subtelomere 18q (top panel) or 13q (lower panel). (C) HCT116 p53-/- and p53+/+ cells were cultured in DMEM with 10% FBS or Hank's buffered saline solution (HBSS) lacking FBS and then assayed by ChIP with either IgG control (grey) or two different p53-specific antibodies Ab6 (red) or FL(black). ChIP was assayed by qPCR with primers proximal to p53 binding sites at 18q, 13q, or p21 promoter. . (D) Doxycyclin (Dox)-inducible p53 H1299 cells were treated without (-) or with (+) Dox in media containing 10% serum or in serum free HBSS, and then subject to ChIP assay with control IgG or p53-specific Ab6 antibody. ChIP DNA was assayed by qPCR using primers specific for p53 binding site at p21 (top panel), p53-negative control IGX1A (middle panel), or p21 positive control (lower panel). Error bars represent SD, and * indicates p value < .05. p-values were determined by Student t-test.