EMBO/sd-character-level-ner huggingface dataset:
Induction of autophagy by a Beclin 1 fragment that disrupts the interaction between endogenous Beclin 1, TAB2 and TAB3.Figure 6(A) Determination of the TAB‐binding domain (TBD) within Beclin 1 (BCN1). Yeast two‐hybrid technology was used to screen for positive (+) or negative interactions (−) between BCN1 fragments and full‐length TAB2. (B) Schematic presentation of the constructs used in subsequent experiments (BBD, Beclin‐binding domain). (C) Confirmation of the TBD by immunoprecipitation. The indicated constructs were transfected into HeLa cells, followed by lysis, immunoprecipitation of HA‐tagged full‐length TAB2 or TAB3 and detection of His‐tagged constructs. (D, E) Inhibition of the interaction between endogenous BCN1, TAB2 and TAB3 by a BCN1 fragment corresponding to the TBD. HeLa cells were transfected with pcDNA3.1 (empty vector) or with plasmids encoding the TBD or a His-tagged Beclin 1 variant lacking the TBD (His-BCN1ΔTBD), followed by co‐immunoprecipitation of endogenous TAB2 (D) or TAB3 (E) and detection of BCN1. (F) Induction of autophagy by the TBD. HeLa cells stably expressing GFP-LC3 were transfected with pcDNA3.1 or constructs encoding the indicated BCN1 variants. After 24 h, Flag‐tagged proteins were detected by immunoblotting and the frequency of cells with >5 GFP-LC3+cytosolic puncta (GFP-LC3VAC cells) was assessed (mean values±s.d., n=3; *P0.01, **P0.001 versus pcDNA3.1‐transfected cells). (G, H) Mechanisms of autophagy induction by the TBD. (G) U2OS cells stably expressing RFP-FYVE were transfected pcDNA3.1 or plasmids for the expression of TBD, BCN1 or BCN1ΔTBD in combination with a control siRNA (siUNR) or siRNAs that effectively deplete VPS4, BCN1 and TAK1. Forty‐eight hours later, the percentage of cells exhibiting FYVE-RFP+ dots (FYVE-RFP+) was assessed. (G). Alternatively, HeLa cells stably expressing GFP-LC3 were transfected with pcDNA3.1 or with a TBD‐encoding plasmids plus siUNR or siRNAs specific the indicated proteins. Forty‐eight hours later, the percentage of GFP-LC3VAC cells was determined (H). Results are mean values±s.d. (n=3, *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells). (I) Inhibition of TBD‐induced autophagy by dominant‐negative (DN) TAK1. Cells were co‐transfected pcDNA3.1 or vectors encoding WT TAK1 (TAK1WT) or a DN TAK1 variant (TAK1K63W) alone or together with a TBD‐encoding plasmid for 24 h, followed by the quantification of GFP-LC3VAC cells (mean values±s.d., n=3; *P0.01 versus pcDNA3.1‐transfected cells).
Two additional hemocyte populations are produced after wasp infestation.Figure 5A) UMAP projection representing the 16 clusters of cells identified in the WI sample. Note the presence of two additional clusters, LMLM-1 and LM-2, compared to the NI sample. The two clusters correspond to the lamellocytes. B) Estimation of the number of cells of each cluster per larva in normal condition (NI, orange) and after wasp infestation (WI, blue). For each cluster, the cell number was deduced from the total number of plasmatocytes and lamellocytes numerated and the proportion of each cluster in the singe cell datasets. C) GO terms enrichment analysis for each cluster of the WI dataset. Represented as in Figure 2C. D) Dot plot representing the expression levels (gradient of red levels) and the percentage of cells (size of the dots) that express the top 5 markers of each cluster in the WI dataset.
Figure 2A In vitro ubiquitination assays of LUBEL-RBR-C WT or catalytically dead C2704A in combination with Ube1 and two different Drosophila E2s, UbcD10 or Effete/UbcD1. Reactions were terminated at indicated times and synthesized Ub chains were detected by immunoblotting using anti-linear Ub antibody. Total protein loading was visualized by Ponceau S staining. * : nonspecific band. B In vitro deubiquitination of Ub chains synthesized by HOIP- and LUBEL-RBR-C. Ub chains produced by RBR-C (Human or Drosophila) with UbcH7, UbcD10 or Effete were incubated with a linear linkage-specific DUB, OTULIN (WT or catalytically dead C129A (C/A) mutant) or a Lys-linkage-specific DUB, vOTU. The DUB-treated samples were subjected to Coomassie staining or immunoblotting using anti-Ub antibody. C Thioester formation assay using Atto647-labelled Ub. Ubiquitination assay using LUBEL-RBR-C WT (left panel) or C2704A (right panel): lanes 1/2 Atto-Ub + E1, lanes 3/4 + ATP, lanes 5/6 + E2, lanes 7/8 + E3, and lanes 9/10 + Ub-His6. In the presence of N-terminally tagged Atto-Ub and C-terminally tagged Ub-His6, only Ub2 product can be synthesized and longer chain formation is restricted. Samples are run without or with DTT in odd or even numbered lanes, respectively. The gels monitor the Atto-labelled Ub. D The linear Ub chain formation activity of LUBEL-LDD mutants. Recombinant proteins of RBR-C WT, C2704A mutant, and LDD mutants (R2754A and D2755A) were assessed for their activity by in vitro ubiquitination assay. The samples were resolved on a gel and stained with Coomassie or immunoblotted using anti-linear Ub antibody. E Linear Ub chain formation by full-length LUBEL transient expression in insect cells. Full-length Myc-LUBEL was transiently expressed in Drosophila Schneider 2 (S2) cells, and Myc-HOIP alone or with HA-HOIL-1L in HEK293T cells. Total cell lysates (TCL) of control and transfected samples were incubated with immobilized GST-Linear-Tandem Ub binding entity (Linear-TUBE) containing three tandem repeats of ABIN-1-UBAN. Pulldown samples were blotted with anti-linear Ub antibody, while TCL were blotted with anti-Myc antibody for exogenous LUBEL and HOIP, anti-HA antibody for HOIL-1L, and anti-Tubulin antibody for loading. Input of GST proteins was analyzed by Ponceau S staining. * : nonspecific band.
Figure 3A Yeast two-hybrid assay of the interaction between ATX1 and SEC. The full-length coding sequence (CDS) of ATX1 was cloned into the bait vector pGBKT7. A full-length SEC CDS and a truncated fragment were cloned into the prey vector pGADT7 to express complete or truncated SEC-N (including only TPR domains at the N-terminus), respectively. AD vectors expressing SEC or SEC-N were each cotransformed with BD-ATX1. SD/2, SD/-Leu/-Trp medium; SD/4, SD/-Ade/-His/-Leu/-Trp medium. Cotransformed pGBKT7-53/pGADT7-T was used as a positive control. B Immunoblot analysis for O-GlcNAcylation of ATX1 by SEC using anti-CTD110.6 antibody. HA-tagged ATX1 was expressed alone or together with Flag-tagged SEC in tobacco leaves. Nuclear proteins were extracted from tobacco mesophyll cells, and expression of FLAG-SEC and ATX1-HA was confirmed by western blotting. C Flowering phenotype of 35S::ATX1-HA overexpression transgenic lines in Col-0 and sec-5 backgrounds, respectively. Scale bar = 1 cm. D Immunoblot analysis using anti-HA antibody to confirm ATX1-HA expression in Col-0 and 35S::ATX1-HA-overexpressing lines ACTIN was used as a loading control. Flowering time phenotype analysis for Col-0, sec-5 and 35S::ATX1-HA transgenic lines Experiments were repeated three times, for each line, a total of 62 plants was counted for statistical analysis of rosette leaf numbers Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01. n.s., not significant. Flowering time phenotype analysis for Col-0, sec-5 and 35S::ATX1-HA transgenic lines The percentages of plants with visible buds and the bolting rates were calculated on day 21 and day 24 after plant germination, respectively. Experiments were repeated three times, for each line, a total of 62 plants was counted for statistical analysis of visible buds percent and bolting rates. Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01. n.s., not significant.
Figure 8Shown are the architecture of CNFY, Pasteurella multocida toxin PMT and two uncharacterized proteins from Pseudomonas syringiae. The released fragment of PMT contains three domains of which C1 is required for membrane binding, the C2 domain has an unknown function and the C3-domain activates heterotrimeric G-proteins by deamidation. The two Pseudomonas syringiae proteins A0A0P9UH04 and A0A0N8SZE6 represent two uncharacterized toxins that encode catalytic domains of the indicated type. While sequence alignments unequi­vocally reveal a CNF-like imperfect β-barrel in PMT, the presence of this domain in the P. syringiae toxins is less obvious.
Figure 2A Basal excitatory synaptic transmission of CA3-CA1 connections in acute hippocampal slices treated with DMSO (0.03%) or the VPS34 inhibitors, VPS34IN1 (3 µM) or SAR405 (20 µM). Representative fEPSP traces (above) and the relative slope of fEPSPs (below) are shown. VPS34 inhibition causes reduced excitatory synaptic transmission down to 46.0 ± 4.1 % and 55.0 ± 4.4 % for VPS34IN1 and SAR405 at the end of 60-min treatment. N = 6 slices per condition from ≥ 4 mice; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001 B Increased paired paired-pulse facilitation (PPF) at hippocampal CA3-CA1 synapses after VPS34 inhibition. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10-500 ms) is plotted below, indicating an increased facilitation of the second response in VPS34IN1-treated acute hippocampal slices. PI(3) P depletion causes a significant facilitation of the second fEPSP, indicative of reduced initial release probability. N = 6 slices per condition from 6 mice; Two-way ANOVA. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001 C Synaptophysin-pHluorin responses normalized to baseline from neurons stimulated with 12 consecutive trains of 20 APs (20 Hz, 1 s) with 30 s gap between each stimulus. Neurons are acutely treated with DMSO (0.1 %) or VPS34 inhibitor1 (50 µM) starting from the onset of 2nd stimulation. The timing of the treatment and the stimulation trains are indicated. D Quantification of data from C. Net difference of Synaptophysin-pHluorin fluorescence 2 s before and 28 s after each stimulation (2 s before the next stimulation) to estimate relative surface retention of SVs. SV endocytosis is blocked within 5 minutes of treatment with VPS34 Inhibitor1, leading to progressive accumulation of Synaptophysin-pHluorin signal. N = 4 or 5 independent measurements per condition; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001
Inhibition of Gli‐dependent BCC tumor cell growth A, B ASZ001 BCC cells were treated with GlaB (5 μM) or DMSO only.Figure 8A, B ASZ001 BCC cells were treated with GlaB (5 μM) or DMSO only. After the indicated times, a trypan blue count was performed to determine (A) the growth rate and (B) the percentage of cell death. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO. C Gli1 mRNA expression levels were determined by qRT-PCR after treatment of ASZ001 BCC cells with GlaB or DMSO only for the indicated times. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO. D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (D) Change of tumor volume during GlaB or vehicle treatment period (18 days). D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (E) Representative flank allografts average volumes (upper panel). D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (F) HE and immunohistochemistry staining of Gli1, Ki67 and TUNEL of allograft tumor samples. Scale bars represent 50 μm for HE, 25 μm for Gli1, 25 μm for Ki67 and 30 μm for TUNEL stainings. (G) Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (F) D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (H) qRT-PCR of Hh and proliferation target mRNA expression levels. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Shown is the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.
a) The CaaX motif of FBXL2 is not required to bind PTPL1.figf5(a) The CaaX motif of FBXL2 is not required to bind PTPL1. HEK293T cells were transfected with either FLAG-tagged wild-type FBXL2 or FLAG-tagged FBXL2(CaaX/SaaX). 24 h post-transfection, cells were collected and whole-cell lysates (WCL) were immunoprecipitated (IP) and immunoblotted as indicated. (c) PTPL1 stimulates the binding of FBXL2 to wild-type p85β, but not p85β(Y655A). HEK293T cells were transfected with GFP-tagged FBXL2 and either FLAG-tagged p85β or FLAG-tagged p85β(Y655A). Where indicated, HA-tagged PTPL1 or HA-tagged PTPL1(C/S) were also transfected. The experiment was performed as described in a. (d) PTPL1 silencing inhibits the FBXL2-p85β interaction. HeLa cells stably expressing FLAG-tagged FBXL2 were transfected with either an siRNA targeting PTPL1 or a non-silencing siRNA (NS). Forty-eight hours post-transfection, cells were collected and whole-cell lysates (WCL) were immunoprecipitated (IP) and immunoblotted as indicated. (e) During a 72-h serum starvation, NHFs were transfected twice with either siRNAs targeting FBXL2 or PTPL1, or a non-silencing siRNA (NS). Cells were subsequently re-stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition. (f) HEK293T cells were transfected twice with either siRNAs targeting FBXL2 or PTPL1, or a non-silencing siRNA (NS). Cells were transfected with p85β(Y655A) and 16 h after cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated. g) During a 48-h serum starvation, U2OS cells stably transfected with a doxycycline-inducible p85β construct were transfected twice with either an siRNA targeting PTPL1 or a non-silencing siRNA (NS). During the last 16 h before collection, p85β expression was stimulated with doxycycline. Cells were subsequently re-stimulated with media containing serum and collected 30 min later. Denatured cell lysates were immunoprecipitated with an anti-FLAG resin and immunoblotted as indicated. The asterisk indicates a non-specific band. Uncropped images of blots are shown in Supplementary Fig. S8.
Characterization of zonal hepatocytes from freshly isolated human hepatocytes (fPHH) A) Schematic of liver architecture and underlying objective of the study.Figure 1A) Schematic of liver architecture and underlying objective of the study. B) Confocal immunofluorescence image of fPHH at 5 days post plating stained with hGK (red), hGS (green), DAPI (blue) and phalloidin (grey). Objective 63x; zoom 2x; scale bar 25 µm. C) Confocal immunofluorescence image of a fPHH monolayer at 5 days post plating, stained with hGK (red), hGS (green), DAPI (blue) and phalloidin (cyan). Arrow point towards a typical hepatocyte subpopulation shown by different colours. Objective 40x; scale bar 25 µm. D) Percentage of zone 1-3 uninfected hepatocytes day 5 post plating (+/- s.d.) of 4 different donors (blue dots). For each donor, >500 cells were characterized for the final percentages. Kruskal Wallis test (p = 0.0002) followed by Dunn's multiple comparisons test where Z1/hGK++/hGS- versus Z2/hGK+/hGS- has a p value of 0.0140 (the other comparisons of Z1/hGK++/hGS- vs Z3/hGK-/hGS+ and Z2/hGK+/hGS- vs Z3/hGK-/hGS+ were non-significant).
Therapeutic efficacy of intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibodies cocktail in Tha-RABV-infected mice.Figure 1A. Rabies virus infection kinetics in the central nervous system of infected mice. Tha-RABV load (copies/µg of RNA) detected in the thoracolumbar spinal cord, in the brainstem plus cerebellum and in the cerebral cortex of infected mice according to different time-points post-infection (n=4 per time-point). nd: not detected. Horizontal lines indicate the median. B. Follow-up of the clinical signs of mice under different treatments. Heatmaps were established based on a progressive 0-7 clinical score scale (0: no apparent changes | 1: ruffled fur | 2: slow movement, hind limb ataxia | 3: apathy | 4: monoplegia | 5: hind limb paralysis, tremors | 6: paralysis, conjunctivitis/ keratitis, urine staining of the haircoat of the perineum | 7: death). Each line represents one animal throughout time. C. Body weight progression of mice under different treatments. Mice were weighted on a daily basis during the treatment administration, and then twice a week up to 100 days post-infection. All the mice were equipped with iPrecio pumps weighting ca. 3.3 g. Each line represents one animal throughout time. D-E. Mouse behavioral testing. The tested animals were non-infected (n=3 mice with iPrecio pump + n=4 age-related mice without iPrecio pump), infected non-treated (n=5), infected and treated at 7 dpi (n=5, mice #9 to #13), and infected and treated at 8 dpi (n=5, mice #24 to #28). (D) Open field test performed at 7 dpi (prodromal phase) and after the remission of clinical signs for the survivors (80 dpi). (E) Rotarod motor performance at 7 dpi (prodromal phase), 8 dpi (symptomatic phase) and after the remission of clinical signs for the survivors (20, 30 and 60 dpi). Horizontal lines indicate the median. Kruskal-Wallis followed by the Dunn' multiple comparisons test (7 and 8 days post-infection) and Mann-Whitney test (20, 30 and 60 days post-infection). The rotarod performance presented a negative correlation (Spearman r = -0.768) with the clinical score at 8 dpi. *P<0.05, **P<0.01, ***P<0.001. Treatments description= NI: non-infected (n=5); NT: infected, non-treated (n=5); infected, treated at 6 dpi (2+2 mg/kg) (n=4); infected, treated at 7 dpi (2+2 mg/kg) (n=9); infected, treated at 8 dpi (2+2 mg/kg) (n=15); infected, treated at 8 dpi (10+10 mg/kg) (n=4).
Figure 6(A) Experimental plan. Mice were intradermally injected with YUMM 1.7 (CD90.1+) cells. When the tumor reached 80-100mm3 size, mice were treated by gavage with glufosinate (20mg/kg) or vehicle for 14 days. Intradermal YUMM 1.7 (CD90.1+) tumor growth over the time (B) in vehicle and glufosinate (20mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total). number of metastatic lesions per lung (C) in vehicle and glufosinate (20mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total). (D) Representative images of metastatic lesions checked by DAB-H stain for CD90.1 positivity in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per lung were analysed. Scale bar: 2mm. Zoomed area scale bar: 50µm. (E, F) FACS quantification of M1-like MHC class IIhigh (E) and MHC class IIlow (F) CD11c+ CD206- TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). (G) Quantification and representative images of CD11c+ cells over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bars: 50 µm. FACS quantification of M2-like MHC class IIhigh CD11c- CD206+ TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). FACS quantification of M2-like MHC class IIlow (I) CD11c- CD206+ TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). (J) Quantification and representative images of CD206+ cells over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bars: 50 µm. FACS quantification of M1-like MHC class IIhigh (K) TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). FACS quantification of M1-like M2-like MHC class IIlow (L) TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). (M) Quantification of MHC-II protein, expressed as Mean Fluorescence Intensity (MFI), on F4/80+ cells, was obtained by flow cytometry (n=6). (N) RT-PCR quantification of M2 (Arg1 and Cxcr4) and M1 (Cxcl9 and Nos2) markers in vehicle and glufosinate (20mg/kg) treated mice (n=6).
LC-MS/MS-based identifications.Figure 2(A) Bar charts visualising the number of unique identified and quantified peptides, protein groups and gene groups (full proteomes), as well as kinase gene groups (Kinobeads) across the CRC65 cell line panel (n=65 cell lines). Insets indicate the corresponding number of identifications across the entire dataset. (B) Venn diagrams showing the intersection and complements with respect to identifications in the aforementioned categories across both the CRC65 cell line and CPTAC patient dataset (n=89 tumours). Kinase identifications in the CPTAC dataset were extracted from the full proteome data. (C) Same as (A) for the CPTAC dataset. The different proteomic datasets were colour-coded (green=Kinobeads, blue=CRC65 full proteomes and purple=CPTAC full proteomes).
Figure 6Chemical structures of acidic-GSEC modulator (GSM I) and imidazole-based-GSM II and GSM III (GSM B in (Szaruga et al, 2017)). Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from vehicle (DMSO) or GSM-treated (1 µM final concentration) MEF cells co-expressing respective WT or mutant NCT GSEC with APPC99 were determined by ELISA. Data generated using 0.3 µM GSM-treated MEF cells is shown in Figure EV4. Data are presented as mean ± SD, N ≥ 3 independent experiments. Aβ 37/40 and Aβ 38/42 ratios were calculated to evaluate the efficiency of the GSEC-mediated cleavage of APPC99 (Figure EV4) and statistical analysis (P values) (two-way ANOVA with Sidak's post hoc test) for the effect of GSMs on WT and mutant complexes is presented (response to GSM normalized to the corresponding vehicle condition).
Elevated sphinganine level causes genomic and epigenomic aberrations leading to senescence and ageing signatures in human cardiomyocytes: Sphinganine induces DNA damage in human cardiomyocytes depicted here (A-C) by γH2A.Figure 2Sphinganine induces DNA damage in human cardiomyocytes depicted here (A-C) by γH2A.X+ nuclei in cardiac-specific ACTN2+ cells (n = 3 biological replicates; # of cells quantified = 147 per condition) Sphinganine induces DNA damage in human cardiomyocytes depicted here (D) by neutral comet assay of DHS treated hCMs. (E) Quantification of comet length in the indicated conditions. Elevated DHS levels lead to ageing signatures in hCMs shown here by (F-G) SA-β-GAL activity, Respective graphs depict the quantification of each of the ageing signatures are on the right. Elevated DHS levels lead to ageing signatures in hCMs shown here by (H) p21 activation (# cells quantified per replicate = 150-200), (I) HP1-α+ nuclear foci (# of cells quantified per replicate = 120-200) and (J) disruption of the nuclear envelope marked by Lamin B1 along with an increase in H4K16ac staining (# of cells quantified per replicate = 100-150 per condition). Respective graphs depict the quantification of each of the ageing signatures are on the right. (K) Proteome analysis confirms sphinganine-induced disruption of (epi) genomic integrity (n = 3). High sphinganine levels cause significant increase in H3K27ac and H3K56ac marks in hCMs (L) and (M) in aged human hearts. (N) Violin plot depicting the distributions of the grey-scale nuclear intensity of the indicated markers. (When not specified, the experiments were conducted in at least 3 biological replicates). Scale bar = 10 µm.