EMBO/sd-character-level-ner huggingface dataset:
Figure 1A The schematics of human NEAT1_2 (WT) and the mutants with deletions in the 3′ terminal regions. The NEAT1 transcripts are shown above with a scale. The gray dashed lines represent the deleted regions. The positions of NEAT1 probes used in SRM (blue) and EM (orange) are shown. B (left) SRM images of paraspeckles in HAP1 NEAT1 ∆3′ mutant cells (∆3′) detected by NEAT1_5′ (green) and NEAT1_15k (magenta) FISH probes in the presence of MG132 (5 μM for 6 h). Scale bar, 500 nm. (right) Graph showing the proportion of paraspeckles with localization of the NEAT1 3′ ends in the core or in the core and shell (n = 44). C (upper) EM observation of the paraspeckles in ∆3′ cells using NEAT1_D2 probes in the presence of MG132 (5 μM for 17 h). Scale bar, 100 nm. (lower, left) Graph showing the proportion of localization of the NEAT1 region detected by NEAT1_D2 probe (248 gold particles) within the paraspeckles in ∆3′ cells. (lower, right) Graph showing the proportion of localization of NEAT1_D2 probes in each paraspeckle in ∆3′ cells (n = 17). The box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top). D SRM images of the paraspeckles in ∆16.6-20.2 kb and ∆20.2-22.6 kb cells detected by NEAT1_5′ (green) and NEAT1_3′ or 19k (magenta) FISH probes in the presence of MG132 (5 μM for 6 h). Scale bar, 500 nm. E Graph showing the proportion of paraspeckles with localization of the NEAT1 3′ ends to the core and shell or the core in ∆16.6-20.2 kb and ∆20.2-22.6 kb cells treated with MG132 (5 μM for 6 h). (∆16.6-20.2 kb: n = 230, ∆20.2-22.6 kb: n = 99) F Schematics of the NEAT1_2 configuration in WT and deletion mutants.
Figure 2A. Schematic drawing of human Timeless and its known domain structure (NTD: N-terminal domain; DBD: DNA-binding domain; PBD: PARP-binding domain). A multiple sequence alignment of vertebrate Timeless sequences is shown underneath, with amino acid conservation coloured according to the Clustal colour scheme. The alignment is annotated with the extent and secondary structure elements of the two helical domains (N-term and C-term) composing the DBD. B. Ribbon drawing of the 1.15 Å crystal structure at of the DBD C-term. Helices are in red and labelled H1 to H4. C. Ribbon drawings of the N-term and C-term domains of the DBD determined by NMR. The two domains are shown in the same orientation to highlight their high degree of three-dimensional similarity. The superposition of the 20 lowest energy structures is shown for each domain. D. The DNA-binding affinity of DBD was measured by fluorescence anisotropy, titrating the DBD protein against Cy3 3'-labelled ssDNA, dsDNA and G4 DNA The top panel shows binding curves for ss- and dsDNA, the bottom panel shows the binding curve for the G4 DNA substrate. E. Ribbon diagram of the superposition of DBD C-term with the highly similar DNA-binding domains of telomeric protein TRF1 (PDB ID 1W0T) (Court et al., 2005) and the bacterial cell-cycle regulator GcrA (PDB ID 5Z7I) in complex with their DNA substrates. A similar DNA-binding mode by DBD would cause a steric overlap of helix H4 with the phosphate backbone of dsDNA. DBD C-term is in light blue, TRF1 and GcrA proteins in brown and their DNA substrates in khaki.
Fig.Figure 5A) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). Nuclei were counterstained with DAPI (blue). MuSCs were cultured alone (-) or with EVs (EVs FAPs) isolated from FAPs in vivo exposed or not to TSA (CTR and TSA) and transfected or not with Antagomir-206 (- and A-206). (n=7 for all samples, but antagomir's samples n=3, biological replicates). Scale bar =50 μm.(C) Graph relative to Notch3 expression in MuSCs isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA), and in EVs derived from FAPs (EVs-FAPs) isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA) (n=3). Star (*) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice (CTR); *p<0,05;. hash (#) means significance compared to EVs-FAPs CTR, ##p<0,01. § indicates significance by Anova test; §§ p>0,01. (D) Graph relative to Notch1 expression in MuSCs isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA), and in EVs derived from FAPs (EVs-FAPs) isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA) n=3. Star (*) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice (CTR); *p<0,05;. hash (#) means significance compared to EVs-FAPs CTR; *p < 0.05. § indicates significance by Anova test; §§ p>0,01. (E) Representative images showing myofibers cultured alone (Ctr) or with extracellular vesicles(F) From left to right: representative images of myofibers cultured for 24 hours alone (-) or with conditioned media of FAPs that were previously exposed or not to TSA and GW4869 (MEDIA-FAPs) or extracellular vesicles (EVs-FAPs) collected from FAPs exposed or not to TSA in vivo (CTR and TSA) and transfected with antagomiR-206 (TSA A-206). Myofibers have been stained for EdU(Cyan), immunofluorescence with Pax7 (green), MyoD (red). Nuclei were counterstained with DAPI (blue). (n=3). Scale bar = 25 μm.
a) Cartoon of the FPP assay illustrating the cellular localization of the proteins and the position of the fluorescent protein tag relative to its respective membrane.figf6(a) Cartoon of the FPP assay illustrating the cellular localization of the proteins and the position of the fluorescent protein tag relative to its respective membrane. GAPDH-RFP is shown in red and CFP-LC3 is shown in green. (b) NRK cells stably expressing CFP-LC3 were transiently transfected to express GAPDH-RFP and subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 100-s treatment with 4 mM trypsin. The inset shows the overlap of the GAPDH-RFP and CFP-LC3 signals (4 × magnification of the central area (outlined by a box) of the micrographs. Scale bar, 10 μm.
Cryo-EM structure of the TRAPPIII-Rab1/Ypt1 nucleotide exchange complex Cryo-EM density map colored by each subunit.Figure 2Cryo-EM density map colored by each subunit. Bottom panel is rotated 90° relative to the top panel.   Colored as in A, the refined atomic model of the TRAPPIII   complex. Network map depicting all of the 108 cross-links found between the TRAPPIII complex subunits and Rab1/Ypt1. Blue lines are indicative of inter-protein cross-links, red lines denote cross-links detected within a single protein. The number of residues in each peptide are labeled within each box and the relative position of the cross-link represents its location within the primary sequence. The fraction of total cross-linking sites observed in each polypeptide. The cross-links mapped onto the refined model. Cross-links are shown as black lines connecting black spheres. A histogram depicting the length of each cross-link that was mapped to the structural model. The distance was measured between the α-carbons of each pair of cross-linked residues. Only one of the cross-links is beyond the maximum theoretical restraint (30Å) of the cross-linking chemical, DSSO.
Figure 2A - HeLa cells were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with L2incDGFP at MOI=0.15. Thirty hours later the cells were disrupted and bacterial titers (IFU=inclusion forming unit) were determined by re-infecting fresh HeLa cells as described in the methods. The mean ± SD of three independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05. B - HeLa cells were transfected with control siRNA or two siRNAs against TG2. Two days later, the efficiency of the silencing was assessed by western blot using anti-TG2 antibodies and anti-actin antibodies as loading control (bottom). Duplicate wells were infected with L2incDGFP and progeny was analyzed as in (A) (top). The mean ± SD of four independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05. C - Same as in (A) except that C. trachomatis serovar L2 (left) or D (right) were grown in primary cells isolated from fallopian tubes. For serovar D, IFU were determined 48 hpi. The mean ± SD of four to five independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05. D - HeLa cells were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with L2incDGFP at MOI=0.15. Thirty hours later the cells were fixed and analyzed by flow cytometry. The percentage of infected cells (left) and the mean fluorescence of the infected population (right) ± SD are shown for three independent experiments. P-values of Student's paired t-test are indicated when <0.05. A representative field for each condition is shown, scale bar = 10 μm. E - Primary epithelial cells isolated from fallopian tubes were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with C. trachomatis serovar L2 (left) or D (right). Twenty-four hours later the cells were fixed, bacteria were stained using FITC-labeled anti-Chlamydia-LPS antibodies, and the mean size of the inclusions manually determined using ImageJ, on twenty inclusions per experiment. The mean ± SD of three independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05. F - Mice were infected intravaginally with 105 IFU of C. muridarum. Twenty-five days later the mice were sacrificed and the upper genital tract, from the uterine horn to the oviduct, was collected. The right part was used for bacterial burden assessment (top left). The left part was rinsed with PBS and observed with a binocular magnifier (right) to determine the hydrosalpinx score (bottom left). Each dot represents one mouse, the mean ± SD is shown. P-values of Mann-Whitney test are indicated when <0.05.
circHIPK3:miR-30c:DLL4 dysregulation affects cell cycle(A, B) qPCR of CCNB1 and CCNE1 levels in scr, circHIPK3 KD and DLL4 KD cells at 0 and 24 hours post induction (n=3).Figure 7(A, B) qPCR of CCNB1 and CCNE1 levels in scr, circHIPK3 KD and DLL4 KD cells at 0 and 24 hours post induction (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (C) Representative plot of cell cycle distribution at 48 hours post induction in scr, circHIPK3 and DLL4 KD cells, with G1 phase (blue), S phase (pink) and G2/M phase (green) highlighted. (D) Analysis of fold change in number of cells in G1, S and G2/M phases of the cell cycle from 0 to 24 hours post-induction (n=4) (i) or 0 to 48 hours post-induction (n=3) (ii). Cells used were scr, circHIPK3 KD or DLL4 KD stable TREx cells and cycle analysis was performed using propidium iodide and flow cytometry (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (E) qPCR for ORF57 DNA levels in TREx cells at 48 hours post lytic induction with treatment of RO 3306, nocodazole or thymidine (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (F) qPCR for ORF57 DNA levels at 48 hours post lytic induction with treatment of RO 3306, nocodazole or thymidine in scr or DLL4 KD cell lines (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.
a, BMDMs were infected with mCherry-expressing M.f3a, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 4 h and immunostained for LC3 (LC3B antibody) or ATG12. b, Quantification of results from a (n = 3 per group, **P  0.001). c, Western blot analysis of LC3 (LC3B antibody) from cell lysates from a. d, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 6 h and immunostained for LAMP1. d, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 6 h and immunostained for LAMP1. e, Quantification of results from d expressed relative to control BMDMs (n = 3 per group, **P  0.001). f, BMDMs were infected with M. tuberculosis and colony-forming units (c.f.u.) at t = 0 and t = 16 h were determined by plating. Results were normalized to t = 0 (n = 3 per group, *P < 0.02). g, RAW264.7 macrophages transduced with lentivirus expressing green fluorescent protein (GFP) or parkin were infected with M. tuberculosis and c.f.u. were determined after 16 h. Results were normalized to GFP-expressing cells (n = 3 per group, **P < 0.0076). h, U937 human macrophages expressing either scrambled or PARK2 shRNAs were infected with M. tuberculosis for 36 h and c.f.u were determined. Results were normalized to t = 0 (N = 3 per group, *P< 0.02). All errors, s.e.m.
Figure 1(A) RAW 264.7 macrophages infected with heat killed (top panels) or live (bottom panels) S. aureus USA300 (in red) were imaged by DIC time-lapse microscopy at 6 h post-infection. The images on the left are of time zero in the image sequence and show the presence of S. aureus. The images on the right are of the same cells and the presence of membrane ruffles and dynamic filopodia are denoted by the yellow arrow heads. Candidate pinosomes are denoted by yellow arrows. (B) RAW 264.7 macrophages expressing the lipid biosensor for PI(4,5) P2, PH-Plcδ-RFP (in red), were left uninfected or were infected with live GFP-expressing S. aureus USA300 (in green) for 8 h prior to fixation. The macrophage plasmalemma and extracellular bacteria are indicated by Cy5-conjugated wheat germ agglutinin (WGA, in blue). As a control, to demonstrate displacement of PH-Plcδ-RFP from the macrophage membrane, transfected cells were treated with 10 µM ionomycin in the presence of 1 mM CaCl2 for 20 min prior to fixation. These images are representative of three independent experiments. (C, D) Internalization of FITC-Dextran by uninfected (top row) and S. aureus (in blue)-infected (bottom row) RAW 264.7 macrophages at 10 h post-infection. These images are representative of three independent experiments. The quantitation of FITC-dextran uptake by either uninfected or infected RAW 264.7 and primary bone marrow derived macrophages is shown in (D). For each experiment, macrophages were treated with 1 mM concanamycin A for 30 min prior to image acquisition. Shown is the mean fluorescence intensity (MFI) in arbitrary units (a.u.) ± standard deviation where each symbol represents the measurement of a single cell. The data derive from three independent experiments and n.s. indicates the data are not significant as determined by an unpaired two-tailed t-test (p>0.05). (E) Potential for co-localization of FITC-dextran (in green) with phagocytosed S. aureus expressing mCherry (in red). The micrographs depict representative macrophages infected with S. aureus at 10 h post-infection and the white arrows point to mCherry positive bacteria that are co-localizing with dextran. Macrophages were treated with 1 μM concanamycin A prior to imaging live.
Figure 6(A Control and GRASP55 KO clones were pre-treated with DMSO or BFA (5μg/ml) for 30 min and SL output monitored by [3H] - sphingosine pulse-chase assay (8 h chase). Total GSL levels were quantified and expressed as fold changes with respect to control (A). B) Control and GRASP55 KO clones were pre-treated with DMSO or BFA (5μg/ml) for 30 min and SL output monitored by [3H] - sphingosine pulse-chase assay (8 h chase). The GM/Gb ratio was calculated and represented (B). (C) HeLa cells were transfected with indicated GCS constructs for 16 hours and SL output monitored by [3H] - sphingosine pulse-chase assay (8 h chase) in the presence or absence of BFA (5μg/ml). Total GSL levels were quantified and expressed as fold changes respect to control. NT refers to non-transfected cells. (D) HeLa cells KO for LCS were transfected with indicated LCS RUSH constructs for 16 hours, and expressed enzymes were retained in the ER (-biotin) or placed in the Golgi by addition of biotin (40 μM) (+biotin) and SL output monitored by [3H] - sphingosine pulse-chase assay. The ratio of GM/Gb is represented. (E) Schematic representation of how the change in localization of GCS in GRASP55 KO with respect to control (pink dashed box vs blue dashed box) from trans- to cis-Golgi results in preferential access to ceramide and thus an increased production of GSLs. (F) CERT was silenced using siRNAs for 72 hours in control and GRASP55 KO (#2) clone before subjecting to [3H] -sphingosine pulse chase assay. GSLs were quantified and represented as fold change with respect to control.
Figure 2Relationship between ANXA1 mRNA expression levels and disease-free survival (DFS) in colon cancer patients. The relationship between ANXA1 mRNA expression levels and DFS was investigated in a database of 466 primary colon carcinomas, assembled by pooling four independent gene-expression array datasets from the NCBI-GEO online repository (GSE14333, GSE17538, GSE31595, GSE37892) The association between ANXA1 expression levels and DFS was tested using Kaplan-Meier survival curves, after patient stratification in groups with high, medium and low ANXA1 expression, using three different methods: (A) based on the median of ANXA1 mRNA expression levels (high 50% vs. low 50%); (B) based on the quartile distribution of ANXA1 mRNA expression levels (high 25% vs. middle 50% vs. low 25%); and (C) based on ANXA1 mRNA expression thresholds calculated using the StepMiner algorithm (low vs. high) Overall, high ANXA1 mRNA expression levels were associated with a statistically significant reduction in DFS (p<0.001, log-rank test), irrespective of the method used for the stratification. Differences in ANXA1 mRNA expression levels did not appear to correlate with differences in each tumor's relative content of epithelial cells in the analyzed biospecimens (i.e. tumor cell density) as revealed by the lack of visual correlations with the epithelial cell marker Desmoplakin (DSP).
Figure 4Splenic cells from C57BL/6 mice were cultured with anti-CD3 antibody and anti-CD28 (0.5 µg/ml) antibodies in the presence of graded doses of hTAPBPL-Ig or equimolar amounts of control Ig (3.75 and 5.63 µg/ml) for 1 day The cells were analyzed for CD69+, cells in CD4 and CD8 T cells. Representative flow cytometric and statistical analyses of the percentages of CD69+, and CD44loCD62Lhi Naïve T cells in CD4 and CD8 T cells. The data are expressed as mean + SD (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with control Ig. Splenic cells from C57BL/6 mice were cultured with anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) antibodies in the presence of graded doses of hTAPBPL-Ig (10 and 15 µg/ml) or equimolar amounts of control Ig (3.75 and 5.63 µg/ml) for 1 day The cells were analyzed for CD69+, cells in CD4 and CD8 T cells. Representative flow cytometric and statistical analyses of the percentages of CD69+, and CD44loCD62Lhi Naïve T cells in CD4 and CD8 T cells. The data are expressed as mean + SD (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with control Ig. Splenic cells from C57BL/6 mice were cultured with anti-CD3 antibody (1 µg/ml) or anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) antibodies in the presence of graded doses of hTAPBPL-Ig (10 and 15 µg/ml) or equimolar amounts of control Ig (3.75 and 5.63 µg/ml) for 1 day or 3 days. The cells were analyzed for CD44hiCD62Llo cells, and CD44loCD62Lhi cells in CD4 and CD8 T cells. Representative flow cytometric and percentages of CD69+, and the numbers (X105) of (F, H, J, L) of CD44hiCD62Llo effector memory T cells and CD44loCD62Lhi Naïve T cells in CD4 and CD8 T cells. The data are expressed as mean + SD (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with control Ig.
Figure 5A. Diagram for mutations in σ28 and tarp. Conserved domains in σ factor are shown as numbering. The region 4 of σ28 was replaced by that from σ70 to obtain the chimeric σ28-R4m, and the sequence of −35 element in tarp was replaced by "TTGACA" in the chimeric tarp-35m. The proposed strength for interaction between σ4/−35 in different σ/promoter pairs is indicated by the thickness of arrow. B. Activities of chimeric σ28-R4m in initiating transcription from tarp-35m, when reconstituted with wild-type (WT) or ZBD mutated RNAP core enzyme. Effects of mutating β′ ZBD on transcriptional activities of σ38-RNAP on osmYp. Effects of mutating β′ ZBD on transcriptional activities of σ38-RNAP on osmYp. Effects of mutating β′ ZBD on transcriptional activities of σ70-RNAP on osmYp.
Figure 1A Bar graph and box-and-whisker plots are presented, which show the allelic proportions of WT FOXL2 mRNA and 402C>G FOXL2 mRNA in AGCT tissues from 46 patients analyzed by pyrosequencing. The box plot represents the minimum value, first quartile, median, third quartile and maximum value of a data set. X-axis indicates mRNAs of WT FOXL2 and 402C>G FOXL2. The whiskers extend to the most extreme data points not considered outliers, and the outliers are represented as dots. Comparisons between groups were performed using Student's t-test, and p values are presented. B The relative abundances of WT and variant FOXL2 mRNA were analyzed in KGN and COV434 cells by pyrosequencing (left graph), allele-specific RT-PCR (middle graph) and real-time RT-PCR (right graph). gDNA was detected as a positive control. The relative abundances of the variant FOXL2 mRNA were normalized to that of WT mRNA (set to 1). FOXL2 mRNA levels detected by real-time RT-PCR were normalized to matching gDNA levels. The pyrosequencing data are presented from two independent experiments. The allele-specific semi-quantitative and real-time RT-PCR data are presented as the mean ± SEM from three independent experiments. The p values were analyzed by unpaired, two-tailed Student's t-test (***p < 0.001). n.d. not detected. C RNA-decay rates of WT and 402C>G FOXL2 mRNAs in KGN cells were determined after treatment with 5 µg/mL ActD for the indicated times. The estimated half-lives of each transcript are presented. The data are presented as the mean ± SEM from three independent experiments.
Figure 3Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information: statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant. B Interaction of MXL-1 with TDPT-1 by yeast two-hybrid assays. The reporter yeast strain L40u was co-transformed with expression vectors encoding LexA DBD-MXL-1 and GAL4 AD-TDPT-1 as indicated. Yeasts carrying the indicated plasmids were grown on a selective plate lacking histidine and containing 10 mM 5-aminotriazole for 4 days. C Structure of TDPT-1. Schematic diagrams of TDPT-1 and human TDP2 are shown. The domains shown are the UBA (red) and Tyrosyl-DNA Phosphodiesterase (TDP; green) domains. The bold line underneath indicates the extent of the deleted region (8 bp) in the km68 mutation. An asterisk indicates a premature stop codon in tdpt-1 caused by the km68 deletion. Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information: statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant.