EMBO/sd-character-level-ner huggingface dataset:
a–d) Starved C38 and IB3-1 cells.figf2(a-d) Starved C38 and IB3-1 cells. (a-c) Confocal microscopy images of beclin 1 (green) and hVps34 (red) (a), calnexin (green) and hVps34 (red) (b), and beclin 1 (green) and HDAC6 (red) (c, left) or hVps34 (green) and HDAC6 (red) (c, right). N, nucleus. Scale bar, 10 μm. Yellow indicates co-localization. (d) Immunoblot of Ambra1, hVps15, hVps34, Atg14L and beclin 1. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (e) Confocal microscopy images of HA (red) and calnexin (green) (top) or hVps34 (red) and calnexin (green) (bottom). Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm. (f) Left: confocal images of LC3-positive dots. N, nucleus. Scale bar, 10 μm. Right: percentage of cells containing more than five LC3-positive dots. At least 60 cells were counted in each experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus cells transfected with the empty vector; ANOVA. (g) Left: electron microscopy of HA-beclin 1-transfected IB3-1 cells shows areas enriched in autophagosomes (arrows). Scale bar, 250 nm. Right: number of autophagosomes per cell; 20 cells were counted in each experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus cells transfected with the empty vector; ANOVA. (h) Immunoblot of HA and LC3. (i) Immunoblot of p62. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (j) Immunoblot of beclin 1 in WT and CftrF508del mice. Representative of seven mice for each group. (k, l) CftrF508del mice (n = 3 per group) were administered intranasally with a lentiviral vector encoding beclin 1 (LV-beclin 1) or GFP (LV-GFP). (k) LC3-positive dots per mm2 of lung tissues. Means ± s.d. for lung tissues from three mice per group. (l) Confocal microscopy micrographs of p62. L, lumen. Representative stainings from at least five airway epithelial areas per mouse. Scale bar, 10 μm.
The MSP domain binds the phosphorylated Phospho-FFAT with an affinity in the micromolar range.Figure 3A-D: Surface Plasmon Resonance analysis of the MSP domain of VAP-A binding onto immobilized unphosphorylated (A), monophosphorylated pS209 (B) and pS213 (C), and bi-phosphorylated pS209+pS213 (D) STARD3 FFAT peptides. Representative sensorgrams resulting from the interaction between the MSP domain of VAP-A injected at different concentrations and the different FFAT peptides are shown in (A), (B left), (C), and (D left). Binding curves display the SPR signal (RU) as a function of time. Some samples were measured twice (concentrations printed in bold). Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted, as well as the bulk effect recorded with buffer only. Steady-state analysis of the interaction between pS209 (B right) and pS209+pS213 (D right) STARD3 FFAT peptide, and the MSP domain of VAP-A. Equilibrium responses (Req) extracted from the left panel in association phase were plotted as a function of the dimeric MSP domain of VAP-A concentration, and fitted with a Langmuir binding model. E: Dissociation constants between peptides corresponding to different kind of FFAT motifs and the MSP domains of VAP-A and VAP-B. The kinetic experiments were performed at 25 °C in Tris-HCl 50 mM, pH 7.5, NaCl 75 mM buffer supplemented with 0.005% (v/v) surfactant polysorbate 20 (P20, GE Healthcare). Mean of n independent experiments: n=2 for pS209 and pS209+pS213; 3 for pS213; and 4 for the non-phosphorylated FFAT. Uncertainties are obtained from the standard deviation considering a t-distribution coefficient for a risk factor of 32%.
Figure 1O-GlcNAcylation signals of TDP-43 were examined by immunoblotting with immunoprecipitated TDP-43 from SH-SY5Y cells. IgG immunoprecipitation served as a negative control. The O-GlcNAcylated TDP-43 was immunoprecipitated by the RL2 antibody and examined by immunoblotting against a TDP-43 antibody. HEK 293T cells were treated with the indicated compounds for 36 h, and the O-GlcNAc levels of TDP-43 were examined. SH-SY5Y cells expressing Flag-TDP-43 constructs were treated with TMG, and the O-GlcNAc levels of TDP-43 were examined. Endogenous (endo.) and exogenous (exo.) TDP-43 were indicated. Cells overexpressing Flag-TDP-43 were treated with increasing doses of UDP-GlcNAc, and the O-GlcNAcylation levels of TDP-43 were detected.   Quantification of (E).   Overexpression of WT OGT, but not catalytic-inactive OGT mutant, increased the O-GlcNAc levels of TDP-43. OGT protein levels were examined by immunoblotting.   Quantification of (G).   Overexpression of WT OGA, but not catalytic-inactive OGA mutant, abated the O-GlcNAc levels of TDP-43. OGA protein levels were examined by immunoblotting. Overexpression of WT OGT, but not catalytic-inactive OGT mutant, increased the O-GlcNAc levels of TDP-43. Quantification of
Figure 7A Malondialdehyde (MDA) content in leaves of 14-d-old DJ and osprr73 plants treated by 180 mM NaCl for 3 d and normal condition. Data represent means ± SD. n = 6, biological replicates. The asterisks indicate the significant difference (***) P ≤ 0.001 by student's t-test. B DAB staining showing the greater ROS accumulation in the leaves of osprr73 mutant treated by 180 mM NaCl treatment for 3 days. Scale bar, 1 cm. C H2O2 concentration was detected by using H2DCFDA in the root tip of DJ and osprr73 mutant plants. Scale bar, 5 mm. -NaCl and +NaCl represented the untreated or treated plants by NaCl respectively. D Quantitative analysis of H2O2 concentration in the root of DJ and osprr73 mutant. Data represent means ± SD. n ≥ 6. The asterisks indicate the significant difference (***) P ≤ 0.001 by student's t-test. E, F Measurement of SOD (Superoxide dismutase) (E) and CAT (Catalase) (F) enzymes activity in shoots of DJ and osprr73 seedlings with or without NaCl stress. Data represent means ± SD. n = 3 technical replicates. Data are representative from two independent biological replicates with similar result. The asterisks indicate the significant difference (*) P ≤ 0.05 by student's t-test.
M45 targets NEMO to autophagosomes.Figure 6(A) NIH-3T3 cells stably expressing Flag-tagged NEMO were mock infected or infected with wt MCMV-GFP or MCMV-GFP-ΔM45 at an MOI of 6. Cells were fixed 6 hpi, and NEMO distribution was analyzed by immunofluorescence. The arrow indicates the region which is shown in higher magnification in the right corner of the picture. (B) NIH-3T3 cells were transfected with Flag-NEMO and M45-HA expressing plasmids as indicated. 24 h later, cells were fixed and double immunofluorescence staining was performed using antibodies against the HA and Flag tags. Regions shown in higher magnification in the left corner of the pictures are indicated by arrows. (C) NIH-3T3 cells stably expressing Flag-NEMO were mock infected or infected with MCMV-M45-HA or MCMV-ΔM45 at an MOI of 7. Seven hpi, cells were fixed and analyzed as described for panel B. (D) NIH-3T3 cells were transfected with expression plasmids for Flag-NEMO and M45-HA or empty vector as indicated. Plasmids encoding GFP-tagged LC3, Rab5, or Rab7 were cotransfected. 24 hours posttransfection cells were fixed and used for anti-Flag immunofluorescence staining.
Figure 4Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of 8-week old SPF, GF or CONV mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm. Images and comparisons of the number of Prox1+ LECs within initial 100 μm portion (C) and VEGFR3 expression (D, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from SPF, GF and CONV mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm. Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum among SPF, GF and CONV mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.
Figure 1A. Schematic representation of the RNF43 protein. SP; signal peptide, PA; protease-associated domain, TM; transmembrane domain, RING; E3 ligase catalytic domain, DIR; Dishevelled-interaction region, SRR; Serine-rich region, HRR; Histidine-rich region, PRR; Proline-rich region. The region in which oncogenic truncations occur (aa 504-563) is indicated. B. β-catenin-mediated reporter activity in HEK293T cells expressing the indicated RNF43 cancer mutants. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown. C. Western blot analysis showing the effect of RNF43 cancer mutants on V5-FZD5 expression in HEK293T cells. Open and closed arrows indicate mature (post-Golgi) and immature (ER-associated) FZD5, respectively. D. Confocal microscopy of surface labeled SNAP-FZD5 in HEK293T cells upon expression of RNF43 WT or R519X. Cells were chased for 30 min. Scale bars represent 10 μm. E. Confocal microscopy of β-catenin localization in HEK293T cells expressing the indicated RNF43 cancer mutants. Scale bars represent 10 μm. F. Western blot analysis of RNF43 WT and R519X for cytoplasmic β-catenin levels. G. β-catenin-mediated reporter activity in HEK293T cells expressing Wnt3a and WT RNF43, oncogenic RNF43 (R519X) or a LOF RNF43 variant (I48T) after o/n treatment with DMSO or the PORCN inhibitor C59 (1 μM).
Figure 1(A) Confocal laser scanning micrographs showing the localization of a functional CDKA;1:mVenus fusion protein in the wildtype (WT) and cartoons on top highlighting the changes in abundance of CDKA;1:mVenus in the nucleus and cytoplasm during the course of meiosis. The region colored in beige represents the cytoplasm, in green the nucleoplasm, and in white the nucleolus. Scale bar: 10 μm. (B) Quantitative analysis of the signal distribution of the nuclear versus cytoplasmic fraction of CDKA;1:mVenus during prophase I of meiosis as revealed by live cell imaging Twenty cells at each time point were used for the analysis. Error bars represent mean ± SD and two biological replicates were performed. (C) Immunolocalization of CDKA;1 (green) and ASY1 (red) on spread chromsomes in leptotene and zygotene of wild-type plants expressing a functional PROCDKA;1:CDKA;1:Strep construct. The last lane shows a magnification of the region marked by the red rectangle. Arrowheads indicate synapsed regions of homologous chromosomes where CDKA;1 is no longer present. Scale bar: 5 μm. (D) Chromosome spread analysis of the wildtype and the hypomorphic cdka;1 mutant CDKA;1T161D. (a, h) zygotene or zygotene-like stages; (b, i) pachytene or pachytene-like stages; (c, j, k) diakinesis or dikinesis-like stages; (d) metaphase I; (e, i, m, n) end of meiosis I with two (e, m) or three (i) pools of chromosomes; (f) metaphase II; (g) tetrad. Red arrowheads indicate the initiated formation of a phragmoplast. White arrowheads depict mitochondria. Scale bars: 10 μm.
Knockdown of EXOSC10 increases RNA foci and repeat RNA transcripts in cells derived from C9orf72 mutation carriers.Figure 4A. In situ hybridization. G4C2 RNA foci were detected in a C9 patient-derived fibroblast but not in a control-derived fibroblast with non-targeting siRNA treatment (left panels, control/si Ct. middle panels, C9/si Ct). Knockdown of EXSOC10 increased RNA foci (right panels, C9/siEXOSC10#5). Nuclei were stained with DAPI (blue). White arrows indicate nuclear RNA foci. The green arrows show cytoplasmic RNA foci. B. Quantification of %cell with G4C2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. C. Quantification of the number of G4C2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. D. Quantification of %cell with C4G2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. E. Quantification of the number of C4G2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. F, G. Increased endogenous repeat RNA expression upon EXOSC10 knockdown in strand specific RT-qPCR. 10 independent knockdown experiments (Case1 N=4, Case2 N=4, Case3 N=2) were performed.
GATE16 inhibits non-canonical inflammasome activation in human myeloid cells.Figure 4(A) LDH and IL-1B release from siRNA-treated primary human Monocyte-Derived Macrophages (hMDMs) infected with S. Typhimurium orgA- (MOI25) for 16 hours. When specified, the caspase-4/5 inhibitor Z-LEVD (25µM) or the NLRP3 inhibitor MCC950 (10µM) were added to the experiments. (B) LDH and IL-1B release from siRNA-treated primary human Monocyte-Derived Macrophages (hMDMs), primed with IFNγ (10UI/mL) and PAM3CSK4 (100ng/mL) and then stimulated with Nigericin (20µM) for 4 hours. When specified, the caspase-4/5 inhibitor Z-LEVD (25µM) or the NLRP3 inhibitor MCC950 (10µM) were added to the experiments. (C, D) LDH and IL-1B release from siRNA-treated WT or GBP1/2/5-/- U937 Monocytic cell line, primed with IFNγ (10UI/mL) and PAM3CSK4 (100ng/mL) and then infected with (C) with S. Typhimurium orgA- (MOI25) for 10 hours or (D) electroporated with 1µg of E. coli LPS for 4 hours. Φ indicates that no LPS electroporation was performed.
Figure 3A. GFAP staining for cerebral astrocytes, the barrel field of the primary somatosensory cortex (S1BF) highlighted with brackets B. Mitochondrial ultrastructure in cardiomyocytes, kidney tubular cells and hepatocytes at P200 as visualized by electron microscopy. Scale bar 1 µm. (C) Average cross-sectional area of mitochondrio (n=3mice/group Bar graphs represent mean±SD. Statistics for graph One-way ANOVA followed by Tukey"s (D number of cristae per mitochondrio (n=3mice/group) Bar graphs represent mean±SD. Statistics for graph ): One-way ANOVA followed by Tukey"s (E) cristae thickness, and (F) average distance between cristae in mitochondria (n=3mice/group) Bar graphs represent mean±SD. Statistics for graph One-way ANOVA followed by Tukey"s
Figure 3A. MITOL was not degraded in mitophagy. HeLa cells stably expressing HA-Parkin were treated with DMSO or CCCP (10 µM) for 48 h and subjected to an IB assay with the indicated antibodies. B. Schematic diagram of EGFP knock-in for N-terminal tagging. MITOL-specific and PITCh-specific sgRNAs expressed from pX330A-MITOL/PITCh (not shown) individually target the MITOL exon 1 locus and the donor vector. This allows for both the cleavage of the genomic locus and the release of the EGFP-containing cassette. MMEJ leads to the repair of double-strand break via the insertion of the EGFP-containing cassette, resulting in endogenously EGFP tagged MITOL. C. Endogenous MITOL translocates to the ER in later phase of mitophagy. EGFP-MITOL knock in HeLa cells were transfected with HA-Parkin and treated with DMSO or CCCP (10 µM) as indicated times. Cells were fixed, permeabilized and subjected to immunofluorescence analysis with the indicated antibodies. Colocalization was quantified by Manders's coefficient. Means ± SEM of more than 10 cells obtained from three independent experiments. For statistical analysis, a one-way ANOVA with Tukey's multiple comparisons test were performed, ****P < 0.0001. Scale bar represents 1 μm. D. MITOL interacts with FKBP38. HeLa cells or HeLa cells stably expressing mCherry-Parkin were transfected with the indicated vectors and treated with DMSO or CCCP (10 µM) for indicated times. Lysates of cells were subjected to an IP-IB assay with the indicated antibodies. E. ER-localized MITOL is transported from the mitochondria, and is not newly synthesized protein. HeLa cells stably expressing HA-Parkin were co-transfected with vectors for MITOL-KikGR and FLAG-FKBP38. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel. Right panel is schematic experimental model for KikGR in this study.
Figure 3Binding was measured by fluorescence polarization using N-terminally TAMRA-labeled monoubiquitin. The dissociation constants for ubiquitin binding to USP4 WT and C311A are 92 ± 21 nM (Clerici et. al, 2014) and 0.60 ± 0.17 nM, respectively. Error bars are s.d. calculated on five measurements per point.
Ectopic progenitor cells originate from apical RG with perturbed cell-intrinsic behavior.Figure 2(A) Hybrid organoids generated from controls mixed with control-EGFP+ or EML1-heKO-EGFP+ cells (day 20±2). Yellow dotted lines indicate VZ areas. (B) Quantification of control-EGFP+ or EML1-heKO-EGFP+ cells within or at the basal side of VZ areas (3 batches, 3 organoids each, significance based on Mann Whitney test, ***P= 0.0001 for "EGFP+ cells at the basal side of VZ areas"). Data information: Data in graphs are represented as means ± SD. Scale bars: (A) 50µm (C) Representative images and scheme of cleavage plan orientation. Upper panel: cells immunostained for pVim (red) and TPX2 (green), counterstained with DAPI, white dotted line indicates cleavage plane orientation, yellow dotted line VZ-surface. (D) Quantification of horizontal, vertical and oblique plane of cell divisions in control and EML1-deficient organoids (day 20±2; 3 batches, 3 organoids each). Data information: Scale bars: 5µm (E) Quantification of primary cilia length in control and EML1-deficient cortical progenitors (* marks statistical significance in relation to Control 1, # in relation to Control 2, 3 biological replicates, 4 areas per sample, significance based on Kruskal-Wallis-test and Dunn's post hoc test, ***/###P= 0.0001). Data information: Data in graphs are represented as means ± SD. Scheme in C was designed using biorender.com. (F) Electron microscopy of primary cilia derived from control or EML1-deficient cortical progenitors in DMSO-control or following EpothiloneD (EpoD) exposure (blue arrow: basal body, yellow arrow: appendages, red arrow: ciliary pockets). Data information: Scale bars: 0,5µm. (G) Quantification of primary cilia length in control and EML1-deficient cortical progenitors with/without EpoD exposure. 3 biological replicates, 4 areas per sample, significance based on two-way ANOVA and Sidak's multiple comparisons post hoc test, P=0.0213. Data information: Data in graphs are represented as means ± SD.
Figure 7The stalling of replication forks is sensed as replication stress and leads to the exposure of ssDNA, which is recognized by RPA. In response to replication stress, the ATR/Chk1 pathway inhibits CDC25 in order to establish the G2/M checkpoint and prevent mitotic entry. In addition, the formation of GMGs is induced upon replication stress in late G2 and prophase nuclei. GMGs represent assemblies of TIAR together with components of the transcription, splicing and replication machineries, possibly reflecting active transcription at sites of stalled replication. TIAR retains CDK1 in GMGs and contributes to CDK1 inhibition during G2/M checkpoint activation. APH, DNA polymerase inhibitor aphidicolin; ATR inhibitor ATRi (ETP-46464); Chk1 inhibitors Gö6976 and UCN-01 (UCN).
Figure 5Western blot images for the expression of YAP, ATF4, and LAT1/Slc7a5 in control or YAP5SA overexpressed C22 airway epithelial cell line. Knockdown of ATF4 was induced by using lentiviral-mediated inducible scramble shRNA (shScramble, control) or shATF4. Quantitative PCR (qPCR) analysis of YAP/TAZ targets (Cyr61, Thbs1, Amotl2), ATF4 and the targets of ATF4 (Trib3, Slc7a5, Slc7a11). Concentration of L-amino acids (L-AA, left) or EAA (right; leucine, isoleucine, valine) in cell lines in (A). Data are presented as mean ± SEM (n=3 biological replicates for each group). *p<0.05, NS=not significant (Student's t-test). Experimental designs for culture and viral transduction for knockdown constructs (shRNA vectors) targeting Atf4 or Slc7a5 and ectopic expression of Atf4 in 3D secretory organoids. Representative H&E images and IF images showing the expression of secretory cell marker CC10, DATP cell marker CLDN4, and AT1 cell marker CAV1 in organoids derived from Lats1/2 dKO secretory cells with shscramble, shAtf4, or shSlc7a5. CC10 (green), CLDN4 (white), CAV1 (white) and DAPI (blue). Quantification of Scgb1a1+ lineage-labeled tdTomato+CC10+ secretory cells (red), tdTomato+CLDN4+ DATP cells (green), and tdTomato+CAV1+ AT1 cells (blue) in (E). Representative H&E images and IF images showing the expression of secretory cell marker CC10, DATP cell marker CLDN4, or AT1 cell marker CAV1 in organoids derived from Scgb1a1 lineage-labeled secretory cells with or without hydroxytamoxifen-induced ATF4 overexpression. CC10 (green), CLDN4 (white), CAV1 (white), and DAPI (blue). Quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells (red), tdTomato+CLDN4+ DATP cells (green), and tdTomato+CAV1+ AT1 cells (blue) in (G).
Replication-transcription relationship Analysis of Rrm3 recruitment in WT and rpb1‐1.Figure 8Analysis of Rrm3 recruitment in WT and rpb1‐1. Rrm3 IP histogram bars in the y‐axis show the average signal log2 ratio of loci enriched in the immunoprecipitated fraction along the indicated regions in the x‐axis. When the ratio fulfills the P‐value criteria (P 0.01) it is shown in red. Composite profile of Rrm3 occupancy across the average ORF plotted as Rrm3 percentage of significant ChIP hits per segment. Each gene was divided into 10 equivalent segments from the start and end coordinates (segments 2-10). Two additional segments of the same size were considered upstream (5′) and downstream (3′) of each ORF (segments 1 and 12, respectively). The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted. Composite profile of Rrm3 occupancy across the average ARS plotted as Rrm3 percentage of significant ChIP hits per segment. Each ARS was divided into 11 equivalent segments from the start and end coordinates. The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted. Model for a role of RNAPII as a source of genetic instability. Conflicts between transcription and replication machineries affect replication progression causing DSBs at high levels under exogenous replicative stress, which demands the HR machinery for the repair and restart of RFs. RNAPII would have a function facilitating RF progression upon a transcription-replication encounter. This function would be affected in the rpb1 and rpb9 mutants analyzed here, causing RF progression impairment and genome instability.