EMBO/sd-character-level-ner huggingface dataset:
Figure 1A Growth curves show proliferation reduction in ATRX depleted U87 cells. Data are expressed as means ± standard deviation (s.d.), N = 2. 500 cells/well were plated. B β-gal staining revealed the increased senescent cell population in ATRX-deleted U87 cells. Upper panel, micrographs of control and ATRX-deleted U87 cells processed for ATRX immunofluorescence (IF) and β-gal senescence assay. Scale bar, 200 µm. D, DAPI. Lower panel, percentage of senescent cells. Data are expressed as means ± s.d., N = 3, unpaired t test. C, D PML/TelG immuno-FISH (C) shows increased APB formation in ATRX-deleted U87 cells (D). Scale bar, 10 µm. (D) Percentages of APB positive cells are expressed as means ± s.d., N = 3, unpaired t test. E C-circle assays revealed enhanced C-circles formation in ATRX-deleted U87 cells. Reactions lacking DNA or ɸ29 serve as negative controls. 30 ng of indicated template DNA was used in each reaction. 53BP1/TelG immuno-FISH (F) G 53BP1/TelG immuno-FISH shows increased TIF formation in ATRX-deleted U87 cells (G). Arrows denote telomeres positive for 53BP1 signals. Scale bar, 10 µm. (G) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test. H Growth curves revealed increase of cell proliferation in ATRX-deleted U87 cells (A-1 and A-2) after hTERT transduction. Data are expressed as means ± s.d., N = 2. I Western blots show depletion of ATRX protein expression in hTERT-transduced and ATRX-deleted A-1 and A-2 U87 cells.
Figure 4(A) Representative images of NMJ synaptic terminals in control (DVGlut-Gal4 > UAS-HA-dNmnat) and hiw mutant (hiwND9,DVGlut-Gal4 > UAS-HA-dNmnat) larvae. NMJs at larval muscle 4 were identified using HRP (blue in top panel) immunostaining. Insets highlight active zones within single boutons, which were identified by staining for the presynaptic active zone protein Brp (red). An anti-HA antibody was used to stain for an HA-tagged dNmnat (green) transgene to measure protein levels in control and hiw mutant active zones. (B) Quantification of HA-dNmnat levels across the entire NMJ synaptic terminal, normalized to total terminal area and presented as fold change over WT. [WT > HA-dNmnat n = 10 terminals, hiw > HA-dNmnat n = 10 terminals. Unpaired T-test, DF = 18, p = 0.0028 (**)]. (C) Quantification of HA-dNmnat levels per unit area in WT and hiw for synaptic terminals excluding active zones (herein referred to as "terminals") versus active zones (AZs) only. [WT n = 10 terminals, hiw n = 9 terminals, each parsed into terminal minus AZs and AZs only. 2-way ANOVA with Tukey's multiple comparisons, DF = 34, p = 0.0005 for row factor (terminal versus AZs) and p < 0.0001 for column factor (WT vs hiw). WT terminal vs hiw terminal p = 0.9830 (NS). Hiw terminal vs hiw AZs p < 0.0001 (****). WT AZs vs hiw AZs p < 0.0001 (****).]
Behavioral characterization of the Ldb2 KO mice A-H.Figure 2Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Home cage-activity test (A). Voluntary locomotor activities in the home cages were monitored for a week. Data of dark (left) and light (right) phases are indicated. Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Open-field test (B) Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Acoustic startle response (C) Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Prepulse-inhibition test (D) Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in , Morris water-maze test (E) where data in hidden test (escape latency and moving speed, path length and no movement time) and probe test (path length) are shown Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Y-maze test (F). Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Fear-conditioning test (G). Results of contextual (left) and cued (right) tests are shown. Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Hot-plate test (H).
Figure 7(A, B) Immunoblots and immunoprecipitation of cell lysates to analyze endogenous interaction of galectin-9 with TAK1 by human THP-1 cells (A) or mouse peritoneal macrophages (B) left unstimulated or stimulated with AG (1 μg/ml) for 1 h. (C) In vitro glutathione S-transferase (GST) precipitation assay purified histidine (His)-tagged Galectin-9 (+) with GST alone or GST-tagged TAK1. (D) Confocal microscopy of mouse peritoneal macrophages left untreated (NC) (upper row) or stimulated with AG (1 μg/mL) for 2 h (middle row) or infected with H37Rv for 3 h (MOI=5) (bottom row), staining with anti-Galectin-9 and anti-TAK1 antibody. DAPI, nuclei, blue. Scale bar, 5 μm. Data in the right graph show mean + SD of n=12 fields from three independent experiments. The symbols indicate the colocalization ratio of at least 10 cells in each field. (E) Immunoblots of cell lysates to analyze phosphorylated TAK1 by mouse peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 μg/ml) for indicated times; GADPH of cell lysates served as the loading control. Data are representative of at least n=3 independent experiments. (F) Immunoblots of cell lysates of peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 μg/ml) in absence or presence of TAK1 inhibitor 5Z-7-OZ (1 μM) for indicated times. Data are representative of n=3 independent experiments. qPCR analysis of Mmp9 (G) from WT or Galectin-9 KO mouse peritoneal macrophages stimulated with AG (1 μg/ml) for 24h in absence or presence of TAK1 inhibitor 5Z-7-OZ (1 μM). Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 ****, p<0.0001. qPCR analysis of Mmps including Mmp10 (H), Mmp12 (I), and Mmp13 (J) from WT or Galectin-9 KO mouse peritoneal macrophages stimulated with AG (1 μg/ml) for 24h in absence or presence of TAK1 inhibitor 5Z-7-OZ (1 μM). Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 ****, p<0.0001.
Figure 3A. RNA decay assay using synthetic, 5′ radiolabeled pre-miR-1003, immunopurified dmDis3l2 and wild-type (WT) or catalytic mutant (CM) Tailor. Reactions were incubated for the indicated time and separated by 15% denaturing polyacrylamide gel electrophoresis and subjected to phosphorimaging.B. Quantification of at least three independent replicates of experiment shown in A. Data represent mean SD. Half-life was determined by fitting data to single exponential (dmDis3l2 and dmDis3l2/TailorCM) or sigmoidal (dmDis3l2/TailorWT) reaction kinetics. C. RNA decay assay using synthetic, 5′ radiolabeled pre-miR-1003 containing the indicated number or uridine residues at the 3′ end and immunopurified dmDis3l2.D. Quantification of three independent replicates of experiment shown in C. Data represent mean SD. Data were fit to single exponential reaction kinetics.E. Reaction kinetics (kobs) determined in D plotted against number of 3′ terminal uridine residues. Error of fit is shown as SEM. F. Two-step-model for the regulation of mirtron hairpin maturation in Drosophila. Mirtron hairpins are generated by splicing and lariat debranching. Upon export from the nucleus, Tailor uridylates mirtron hairpins by recognizing the 3′ guanine, a hallmark of splice-acceptor sequences. Short uridylation masks the 2nt 3′ overhang required for Dicer-1 to recognize pre-miRNAs. Extended uridine-tails eventually trigger processive, 3′-to-5′ exoribonucleolytic decay by dmDis3l2, resulting in mirtron clearance.
Dynamic interactions between photoreceptors and infiltrating microglia underlying phagocytosis during photoreceptor degeneration.Figure 5Time-lapse confocal imaging (at a rate of one image stack/min) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21-24.Infiltrating microglia established rapid and transient physical contacts with nearby photoreceptorsomata (red and yellow circles) via their processes; these processes often terminated in a "cup"-like structure (arrow) that upon soma contact proceeded to extend across the entire soma (arrowhead, and red circle and *) to engulf it completely. The majority of microglia-photoreceptor contacts were transient with the microglial processes dynamically contacting and releasing photoreceptorsomata in repetitive cycles (e.g. yellow circle and *) (period ≅10-15 min). Time-lapse confocal imaging (at a rate of one image stack/min) was used to document dynamic behavior of ONL microglia in rd10 retinal explants at ages P21-24. Example in which the engulfment of photoreceptorsomata by microglial phagocytic cup was followed up by actual phagocytosis in which the engulfed cell (red *) is translocated intracellularly within the microglial cell toward the cell body. Phagocytosis of soma occurred simultaneously with "probing" of other somata by processes of the same microglial cell (yellow *). Time-lapse confocal imaging (at a rate of one image stack/min) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21-24. Phagocytosis of photoreceptorsomata occurred via flattened microgliallamellipodialmicroglial processes (arrow) that extended across somata (yellow *) to engulf them. Nuclei of phagocytosed photoreceptors within microglialphagosomes occasionally developed staining for propidium iodide (red arrow); these subsequently faded and disappeared over ≅10 min, possibly representing intraphagosomal breakdown. Time-lapse confocal imaging (at a rate of one image stack/min) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21-24. Amoeboid infiltrating microglia lacking extended processes were also observed to phagocytose photoreceptors via phagocytic cups formed at their cell body (red *).
Figure 4(A) Representative confocal images and Quantification of STX within the lysosome in U2-OS and HeLa cells after acute silencing of CLN3 (siCLN3) in DMSO or treated 48h with Tamoxifen. (*** vs siSCR DMSO, °°° vs siCLN3 DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm. (B) Representative confocal image and quantification of Gb3 in ARPE-19 CLN3 KO cells silenced with siRNA against scramble sequence and TFEB (siTFEB) for 72h and treated for the last 48h with DMSO or Tamoxifen. Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm. (C) Representative confocal image and quantification of TFEB in U2-OS cells silenced with siRNA against scramble sequence and CLN3 (siCLN3) for 72h and treated for the last 48h with DMSO or Tamoxifen (5 µM and 10 µM). Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.
Figure 7(A) Import of Mcp3 is dependent on the TIM23 complex. Mitochondria isolated from wild-type and tim23ts cells were incubated with radiolabelled Mcp3 for the indicated time periods. Further treatment and analysis (n=3; SD) was as described for Fig. 5D. (B) Steady state levels of HA-Mcp3 are lower in cells harbouring a temperature sensitive TIM23 allele. Crude mitochondria were obtained at the non-permissive temperature from WT or tim23ts cells containing a plasmid expressing HA-Mcp3. Samples were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, the matrix proteins Mge1 and Yah1 as typical TIM23 substrates, Ugo1 and Fis1 as TIM23-independent substrates. HA-Mcp3 levels were quantified in relation to Fis1 levels. Levels in WT cells were set to 100%. The bar diagram shows the mean with standard deviation of six independent experiments (n=6; SD; **, p < 0.01). (C) Import and processing of Mcp3 is dependent on the mitochondrialmembrane potential. Isolated mitochondria were incubated with radiolabelled Mcp3 or pSu9-DHFR as control for the indicated time periods or for 15 minutes in the presence of either CCCP or o-phenantroline and EDTA (o-Phen./EDTA). After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. L+MPP: radiolabelled proteins were incubated with recombinant MPP. (D) Mcp3 is processed by Imp1/2. Single deletion strains of known mitochondrial proteases and peptidases were transformed with a plasmid expressing HA-Mcp3. Crude mitochondria were isolated and analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins and the HA-epitope. Cox2, a known substrate of IMP; Tom70, a mitochondrial outer membrane protein. (E) C-terminally tagged Mcp3-HA is processed by Imp1. WT and imp1Δ cells were transformed with a plasmid expressing Mcp3-HA and further analysis was performed as described in (D). (F) Mcp3 is processed by Imp1/2 after in vitro import into mitochondria. Mitochondria isolated from WT, imp1Δ or imp2Δ strains were incubated with radiolabelled precursor of Mcp3 or pSu9-DHFR as control. Further treatment and analysis was as described in Fig. 5A. (G) Full-length Mcp3 precursor accumulates during import if IMP processing is abolished. Radiolabelled internally HA-tagged Mcp3 without (HA-Mcp3) or with mutation in the Imp1 cleavage site (D70G+HA) were incubated with mitochondria as in (F). After import samples were incubated with or without PK (20 µg/ml). Samples were analysed by SDS-PAGE and autoradiography. (H) Mitochondrial membrane potential is required for efficient import of Mcp3 precursor into mitochondria. Isolated mitochondria were incubated with radiolabelled Mcp3 in the presence or absence of CCCP. After import mitochondria were incubated with PK as in (G) and analysed by SDS-PAGE and autoradiography. I, 20% of radiolabelled precursor protein used in each import reaction; The arrowhead marks an additional band of the size of the cleaved N-terminus of Mcp3.
Figure 5(A,A') Specific expression of FAM134B mRNA (green) in sensory neurons (red) of P4d vGlut2Cre;R26lsl-tdTommouse. Scale bars, 100μm. The white dotted line indicates the dorsal root ganglia (DRG) where sensory neurons are genetically labeled with Tomato. Insets of (a) and (a') are high magnifications of sensory neurons showing the co-localization of FAM134B mRNA and Tomato. Scale bars, 10 μm. Cultured sensory neurons from DRGs of E14.5 vGlut2Cre;R26lsl-tdTom embryos were infected with same titers of recombinant lentivirus expressing GFP-FAM134B WT (B), G216R(C), S151A-G216R(D), SA-G216R(E), G216R-ΔLIR (F) respectively. Infected sensory neurons (GFP+/Tom+) are indicated with arrowheads. The nuclei of all cells are shown with DAPI staining. Scale bars, 20 μm. Cultured sensory neurons from DRGs of E14.5 vGlut2Cre;R26lsl-tdTom embryos were infected with same titers of recombinant lentivirus expressing G216R supplemented with 5μM KN93 (G) Infected sensory neurons (GFP+/Tom+) are indicated with arrowheads. The nuclei of all cells are shown with DAPI staining. Scale bars, 20 μm. (H) Quantification of the viability of sensory neuron after 48h, 72h and 96h of infection. Sensory neuron viability was calculated as the percentage of transfected sensory neuron (GFP+/Tom+) in total sensory neurons (Tom+). For each time point, four biological replicates were included for statistical analysis. *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA, error bars indicate SEM. "ns" is the abbreviation for "not significant". For all of the groups, at least 10 fields of the slides were included.
Workflow of ligand-detected NMR approach 29 His-tagged enzymes of E.Figure 229 His-tagged enzymes of E. coli central metabolism were mixed individually with four metabolite mixes and T1rho 1D 1H NMR spectra were recorded for every protein-mix combination. All possible interactions between enzymes and metabolites were quantified using the relaxation factor and are displayed in a protein-metabolite interaction map.
Figure 5(A) Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes, treated with 100 nM BafA1 for 9h. n=18 cells/treatment; 3 independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ** P<0.005, *** P<0.0001. (B) WT and Canx-/- MEFs were untreated or treated with BafA1 (10 μM) for 6 h, lysed and analysed by western blot with indicated antibodies. Filamin and β-actin were used as loading control. Dashed line indicates that unnecessary lanes were removed. Western blot is representative of 3 independent experiments. (C) MEF cell lines lacking the indicated genes were treated for 12h with 50 nM BafA1 fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). CST was added where indicated. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph on the right shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=8, 8, 12, 8, 8, 8 cells respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. *** P<0.0001.
Figure 6(A) Venn diagram showing the differentially expressed protein counts between EV-A71-infected and uninfected cells after data mining. (B) Pathway-based classification of proteins present in the infected eluates. (C) Viral titers in siRNA-treated NSC-34 cells. Cells were transfected with various concentrations of siRNA specific to the indicated target genes. The efficiency of siRNA-mediated knockdown at 48h.p.t. was validated by Western blot while cytotoxicity was determined by AlamarBlue assay siRNA- or siNTC-transfected cells were infected with EV-A71 S41 at MOI 10, and the viral titers were determined at 48h.p.i. (D) Drug treatment. EV-A71-infected NSC-34 cells were treated with the indicated drugs, and the viral titers were determined at 48h.p.i.
Figure 3(A) The number of produced exophers positively correlates with the number of in utero embryos. The violet line is a linear regression line, and each orange point represents one animal. All animals were 1-3 days old; n = 54; N = 3. (B) RNAi knockdown of genes regulating the egg laying rate and their presence in the uterus influences exopher production. n = 50-60; N = 2. (C) Embryos retention in the uterus caused by starvation during worm's reproductive period increases exopher production. n = 81-90; N = 3. (D) Schematic representation of the experimental setup for investigating the influence of embryo-conditioned buffer on exopher production. (E) Exposure of young-adult worms to embryo-derived substances increases exopher production. n = 48 and 56; N = 3. (F) Increased eggshell permeability caused by emb-27 knockdown elevates exopher production. n = 103 and 89; N = 3.
GluN2A(N615S) containing NMDAR expression in vitro and in vivo.Fig. 1a The position of N615 in the membrane segment M2 is depicted together with the three channel-forming trans-membrane segments M1, 3, and 4. b In HEK293 cells, recombinantly expressed GluN1/GluN2A and GluN1/GluN2A(N615S) channels were activated by fast glutamate application (1 mM; in the continuous presence of the co-agonist glycine, 10 µM) at holding potentials from -100 to +40 mV in different extracellular Mg2+ concentrations. NMDAR-mediated peak currents were normalized to those obtained at +40 mV. Data points represent mean ± SEM for n = 4-7 different HEK293 cells. Representative current traces evoked in 0 mM Mg2+ at - 60 mV, with 20 and 600 ms applications, are shown below the IV plots and were used to determine the current kinetics (Supplementary Table 1). c Schematic view of the A to G replacement in exon 10 of the mouse Grin2a gene. d Reverse transcription PCR (RT-PCR)-sequence analyses of total brain mRNA show the A-to-G mutation in pos. 0 and two diagnostic silent mutations at pos. -11 and -14 in the pore loop encoding gene segment in Grin2a+/+, Grin2aS/S, Grin2a+/S mice. In Grin2a+/S mice, the overlay of two different colored "nucleotide" peaks, at position 0, -11, and -14 indicate equimolar amounts of mRNA from the Grin2a+ and the targeted Grin2aS alleles. e Immunoblots of forebrain protein lysates of 4-week-old mice (Supplementary Fig. 2) indicate no genotype-specific differences of GluN1, GluN2A, and the AMPAR subunit GluA1 expression relative to the β-actin levels (p > 0.05). The levels of PSD95 and αCaMKII (in its phosphorylated state, pCaMKII) are also comparable between genotypes relative to the GAPDH expression. The GluN2B expression level in the membrane fraction was significantly increased in Grin2aS/S mice when compared to Grin2a+/+ and Grin2a+/S mice but not in the levels of total protein lysates (for statistics: Supplementary Statistics to Fig. 1). The number of mice is given in brackets. Error bars represent mean ± SEM.
Figure 5A, Diagram illustrating the Golgi recruitment mechanisms of PI4KIIIβ. B. Representative western blot for ACBD3 in control and NPC1I1061T patient fibroblasts. Protein levels were normalized to β-actin. Control: n=26; NPC1I1061T: n=27. A paired t test was used for the statistical analysis with *P < 0.05. C. Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for ACBD3. Black and orange squares show expanded views of anti-ACBD3 in Golgi and endomembrane compartments, respectively. D. Analyses of ACBD3 intensity distributed in Golgi (left graph; Control: n=9; NPC1I1061T: n=9) or in endomembranes (right graph; Control: n=1360; NPC1I1061T: n=1645 from 15 cells), normalized to the cytoplasm. E. Confocal images of control (1st column) and NPC1I1061T fibroblasts (2nd column) expressing mCherry-P4M or control fibroblasts expressing P4M-mCherry and GFP-ACBD3 (3rd and 4th columns). F. Quantification of Golgi mCherry-P4M intensity relative to cytoplasm. Control: n=11; NPC1I1061T: n=14; Control + ACBD3: n=12. G. Confocal micrographs from control or GFP-ACBD3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ. Bottom panels show enlarged views of ACBD3, PI4KIIα, or PI4KIIIβ at Golgi regions. H. Quantification of PI4KIIα (left; control: n=42; ACBD3: n=28), or PI4KIIIβ (right; control: n=39; ACBD3: n=20) intensity relative to cytoplasm. I. Confocal images of control and sgACBD3 HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in Golgi and in endomembrane compartments. J. Quantitative analyses of P4M-YFP mean fluorescence intensity at Golgi (left graph) or endomembranes (right graph) normalized to the cytoplasm. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups. For left graph; Control: n=19; U18: n=13; sgPI4K3B: n=12; sgPI4K3B+U18 : n=11; sgPI4K2A + sgPI4K3B: n=12; sgPI4K2A + sgPI4K3B +U18: n= 12; sgACBD3: n=15; sgACBD3+U18: n=16. For right graph; control: n=149; U18: n=251; sgACBD3: n=138; sgACBD3+U18: n=190. Please note that 'control' and 'U18' datasets in Fig 5J are identical to those in Fig. 4C. Data for all CRISPR experiments were collected on the same days.