EMBO/sd-character-level-ner huggingface dataset:
Figure 2RT-PCR analysis in P30 mice showing the presence of Clrn2 transcripts in the inner ear, eye, but not in brain or muscle. β-actin was used as a positive control. Clrn2 transcripts could be detected in both inner (IHCs) and outer (OHCs) hair cells of P15 wild-type mice. Otoferlin (Otof) and oncomodulin (Ocm) transcripts were used as positive controls for IHCs and OHCs, respectively. Ocm transcripts were only present in the OHC lysate, demonstrating that the IHC sample had not been contaminated with OHCs. Auditory phenotyping of clarinet mice at P16 (C), P21 (D) ABR threshold measurements show that Clrn2clarinet/clarinet mice (red) exhibit a severe-to-profound hearing loss affecting all frequencies tested. At 16 kHz in Clrn2clarinet/clarinet mice, the mean ABR hearing thresholds vary from 55-65 dB SPL at P16, 60-90 dB SPL at P21, and to 80-100 dB SPL at P28 and P42. Age-matched Clrn2+/+ (black) and Clrn2clarinet/+ (grey) controls display thresholds within the expected range (15-45 dB SPL) at all frequencies and time-points tested. At P16, all eight Clrn2clarinet/clarinet mice exhibited recordable ABR responses for each frequency tested and the click stimulus. For the longitudinal ABR study, at P21 and P28 three of the seven Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least one frequency-specific/click stimulus. By P42, five of the Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least two frequency-specific/click stimuli. ABR data shown are mean ± s.d. ***p<0.001, one-way ANOVA. Auditory phenotyping of clarinet mice at P28 (E) and P42 (F). ABR threshold measurements show that Clrn2clarinet/clarinet mice (red) exhibit a severe-to-profound hearing loss affecting all frequencies tested. At 16 kHz in Clrn2clarinet/clarinet mice, the mean ABR hearing thresholds vary from 55-65 dB SPL at P16, 60-90 dB SPL at P21, and to 80-100 dB SPL at P28 and P42. Age-matched Clrn2+/+ (black) and Clrn2clarinet/+ (grey) controls display thresholds within the expected range (15-45 dB SPL) at all frequencies and time-points tested. At P16, all eight Clrn2clarinet/clarinet mice exhibited recordable ABR responses for each frequency tested and the click stimulus. For the longitudinal ABR study, at P21 and P28 three of the seven Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least one frequency-specific/click stimulus. By P42, five of the Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least two frequency-specific/click stimuli. ABR data shown are mean ± s.d. ***p<0.001, one-way ANOVA. Averaged DPOAE responses for clarinet mice at P28, showing significantly reduced responses in Clrn2clarinet/clarinet mutants at all frequencies tested. DPOAE data shown are mean ± s.d. *p<0.02, **p<0.01, one-way ANOVA. Please see Appendix supplemental table 1 for exact P-values.
Two Nup50 paralogues in mouse cells (A) Mouse 3T3-NIH cells were transfected with 20 nM control, Nup50A, Nup50B or a combination of Nup50A and Nup50B siRNA.Figure 2(A) Mouse 3T3-NIH cells were transfected with 20 nM control, Nup50A, Nup50B or a combination of Nup50A and Nup50B siRNA. After 72h cells were fixed and stained with mAB414 (green) and antibodies against mouse Nup50 (red). Chromatin is stained with DAPI (blue). The merge of the three channels is shown on the left column. Scale bar: 50 µm. An insert shows a zoom on a representative nucleus for each picture. Scale bar: 10 µm. (B) Quantitation of the mAB414 rim intensity from n=4 independent experiments done as in (A), each represented by a different color. The mean of each individual experiment and condition is represented by a diamond, the mean of all four experiments by a horizontal black line. P-values have been calculated from a paired ratio t-test comparing the mean between the experimental conditions. Error bars show the standard deviations of the means. (C) Western blot showing the amount of Nup50A (top row) for each of the experimental condition. The actin (lower row) is shown as a loading control.
Figure 3A HA‐Flag‐TBC1D5 expression was induced in T‐REx HeLa cells for 20 h, lysates were subjected to GSTpull‐down, −AP2M1. Co‐precipitatedTBC1D5 was detected with anti−Flag antibody. B Control cells and TBC1D5 (shRNA#1) U2OS cell lysates subjected to SDS-PAGE, subsequently blotted with endogenous antibodies for AP2 subunits AP2A1 and AP2M1. C Immunofluorescent staining of AP2A1 in U2OS shRNA control, or shRNA#1 TBC1D5 cells treated with KU0063974 or DMSO (6 h). D U2OS cells stably expressing HA‐Flag‐ATG9A were fixed and stained for endogenous AP2A1 and TBC1D5. Regions of co‐localization are indicated with arrows. E U2OS cells stably expressing HA‐Flag‐ATG9A were starved in EBSS (4 h) or treated with KU0063794 (6 h), subsequently fixed and stained with anti‐AP2A1 antibody. F U2OS cells stably expressing mCherry‐ATG9A were treated with KU0063794 (6 h), lysed in co‐immunoprecipitation buffer. Lysates from DMSO‐ or KU0063794‐treated cells were split and equal volumes were incubated with RFP‐Trap beads or GFP‐Trap beads, or agarose as a negative control, overnight at 4°C. Precipitated proteins were analyzed by SDS-PAGE.
Modulation of microtubule growth by cell spreading and centrosomal actin.Figure 6Schematic illustration of our model according to which cell spreading sequesters monomeric actin to the cortex and thereby enables the centrosome to grow more microtubules. Drawings show top (top line) and side views (bottom line) of cells with increased spreading from left to right. Actin filaments are in green, microtubules in red. RPE1 cells stably expressing centrin1-GFP were plated for 3 h on coverslips coated with different ratios (100:0; 50:50 or 1:99) of fibronectin and PLL-PEG prior to fixation and staining for F-actin (top line and magnified views around centrosome below. Scale bars: 10 µm and 2 µm, respectively) and α-tubulin (bottom line. Scale bar: 10 µm). IIA1.6 B lymphoma cells were plated for 60 min on poly-L-lysine, fibronectin or ICAM-1 coated cover slides prior to be fixed and stained for F-actin (top line) and α-tubulin (bottom line). Scale bar: 3 µm. Quantification of the area occupied by RPE1 cells on the substrate (top left), F-actin content at the centrosome (top right) polymerized tubulin at the centrosome (bottom left) and in the entire cell (bottom right) for the three conditions of cell adhesion described in (B). Measurements came from 3 independent experiments with more than 60 analyzed cells in each. Errors bars represent standard deviations. F-actin and microtubule contents were compared using Mann-Whitney test, and variations of the cell area were compared using unpaired t-test. Quantification of the area occupied by B lymphoma cells on the substrate (top left), F-actin content at the centrosome (top right) polymerized tubulin at the centrosome (bottom left) and in the entire cell (bottom right) for the three conditions of cell adhesion described in (D). Measurements came from 3 independent experiments with more than 80 analyzed cells in each. Errors bars represent standard deviations. F-actin and microtubule contents were compared using Mann-Whitney test, and variations of the cell area were compared using unpaired t-test.
Fig.Figure 5a-b, siRNA-mediated down regulation of CDC components in WT NPCs causes longer cilia (Ofd1 ***p<0.001, Nde1 **p<0.01), retarded cilia disassembly as shown by increased numbers of ciliated cells (Ofd1 **p<0.01, Nde1 *p<0.05) and differentiation of NPCs into TUJ1 positive cells (Ofd1 **p<0.001, Nde1 *p<0.01). In controls, a non-targeting siRNA (scrambled) is used. Western blots at right validate siRNAs-mediated Nde1 and OFD1 depletion. Error bars are s. e. m. Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 3 independent experiments. 275 cells for scrambled, 423 cells for Ofd1 siRNA and 255 cells for Nde1 siRNA. a-b, siRNA-mediated down regulation of CDC components in WT NPCs causes longer cilia (Ofd1 ***p<0.001, Nde1 **p<0.01), retarded cilia disassembly as shown by increased numbers of ciliated cells (Ofd1 **p<0.01, Nde1 *p<0.05) and differentiation of NPCs into TUJ1 positive cells (Ofd1 **p<0.001, Nde1 *p<0.01). In controls, a non-targeting siRNA (scrambled) is used. Western blots at right validate siRNAs-mediated Nde1 and OFD1 depletion. Error bars are s. e. m. Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 3 independent experiments. 275 cells for scrambled, 423 cells for Ofd1 siRNA and 255 cells for Nde1 siRNA. c, VX680 (0.5 μM, a concentration that will specifically allow neuronal differentiation) was used to inhibit Aurora A activity. Bar diagrams below show quantifications. Compared to DMSO-treated control NPCs, a higher proportion of VX680 treated NPCs are ciliated (***p<0.001), exhibiting longer cilia (***p<0.001) and show Tuj1 positive neurons (***p<0.001). Error bars are s. e. m. For ciliated cells and cilium length: n = 3 independent experiments. 164 cells for DMSO-treated and 77 cells for VX680-treated. For NPC differentiation: n = 4 independent experiments. 286 cells for DMSO-treated and 118 cells for VX680-treated. Scale bar 1 μm. d, Cell cycle progression of siRNA or VX680 treated WT NPCs compared to control assessed by EdU incorporation for 24 hours. Inhibition of Ofd1 (*p<0.05), Nde1 (**p<0.01) and Aurora A (***p<0.001) caused significantly reduced incorporation of EdU. e-g, Western blots show that CPAP targeting siRNA treatment depletes endogenous CPAP. Both low -and high -exposures are given (e). Increased cilia length and NPC differentiation upon expression of RNAi-resistant CPAP E1235V compared to WT CPAP (f-g). Error bars are s. e. m. *** p<0.001 (cilia length), * p<0.05 (differentiation), t-test. For cilia length: n = 4 independent experiments. 25 cells for CPAP, 47 cells for CPAP E1235V. For differentiation: n = 4 independent experiments. 145 for CPAP and 81 for CPAP E1235V. Scale bar 1 μm.
JARID2 LMW is a cleaved product of full length JARID2 isofrom-1.Figure 2A schematic showing three different isoforms of human JARID2, as predicted by ensembl annotations, is shown. The translation start site of each isoform is labelled using green arrow. Predicted sizes (140kDa, 120kDa and 106kDa) of proteins corresponding to three isoforms have been indicated. CAGE-seq data showing the transcription start sites of JARID2 in different cell lines. CAGE peak is mainly observed at JARID2 isoform-1 in most cell types. JARID2 gene structure is shown at the top. qPCR measurements of RNA levels (n=3) for the three isoforms of JARID2 in HaCaT cells. The RNA levels are plotted relative to 18S rRNA. Level of JARID2 isoform-1 is significantly higher than that of isoform-2 (***P<0.001) and isoform-3 (****P<0.0001). Agarose gel showing RT-PCR products amplified using primers in exon3 and exon15 (2.9kb) as well as RT-PCR product corresponding to JARID2 isoform-1 (3.7kb) amplified using the first (exon 1) and the last exon (exon18). Only one product at the right size is observed indicating that it is unlikely that JARID2 might be a product of an alternatively spliced isoform of JARID2. Ribosomal profiling data mapped on JARID2 isoform-1 and isoform-2. Main translation site corresponding to isoform-1 is highlighted using a dashed box. CRISPR-Cas9 knockout of JARID2 using a sgRNA guide designed to target main translation start site as seen in (E). Immunoblot revealed that ~80kDa form was removed from JARID2 KO lines. Immunoblot after a full length JARID2 isoform-1 was expressed from an exogenous vector in HaCaT cells showed an increase (2-3 fold) in ~80kDa level. Immunoprecipitation using anti-JARID2 antibody and followed by mass spectrometry identification of the ~80kDa band detected JARID2 peptides spanning only C-terminus of JARID2. The identified peptides are shown as red arrows on schematic of JARID2 sequence.
TRAIL induces cytoprotective autophagy in breast epithelial cells.Figure 1(A) MCF10A-eGFP-LC3 cells were left untreated (Control) or treated with 5 μM rapamycin (Rapa), 500 ng/ml TRAIL or 10 ng/ml TNF for 24 h. When indicated, 10 mM 3‐MA or 5 μg/ml TRAIL‐R2 antagonist antibody was added 1 h before the drugs. Representative confocal images (20 μm scale bars) and the percentages of cells with LC3 translocation are shown. (B) MCF10A-eGFP-LC3 cells were transfected with the indicated siRNAs for 48 h and analysed by immunoblotting (left), or stimulated with 500 ng/ml TRAIL for 24 h and analysed for LC3 translocation (right). (C) MCF10A cells left untreated (Control) or treated with 500 ng/ml TRAIL or 5 μM rapamycin (Rapa) for 24 h were analysed for LC3‐I, LC3‐II and tubulin by immunoblotting. Pepstatin A and E64d were added to the cells 4 h before harvesting. Similar results were obtained in two independent experiments. (D) MCF10A cells were treated with 5 μM rapamycin (Rapa) or indicated concentrations of TRAIL for 24 h, and the increase in the degradation of long‐lived proteins as compared with the untreated cultures (Control) was measured. (E) MCF10A-eGFP-LC3 cells transfected with the indicated siRNAs were left untreated 48 h later (Control) or treated with 500 ng/ml TRAIL for 24 h and analysed for the DNA content by flow cytometry. The percentage of apoptotic cells with sub‐G1 DNA content is shown. The values represent mean±s.d. for three (A, D) or four (B, E) independent experiments. *P‐value 0.05, **P‐value 0.01 and ***P‐value 0.001 as compared with control sample (A-left panel and D), with TRAIL‐treated cells without anti‐TRAIL‐R2 (A-right panel) or with TRAIL‐treated cells without siRNA (B, E).
Fig.Figure 3Cytokine levels in plasma of individual mice obtained from blood at 7 weeks after vector injection. The distinct symbols used for each individual mouse are identical to the ones used in Fig. 2. Mean values ± SD of n=2 technical replicates. Immunohistochemistry of paraffin embedded sections from the lungs, brain meninges, brain striatum, spleen and liver of the vector-injected mouse M16 (CAR) and a control mouse injected with PBS (PBS) stained against CD8 (αCD8), the CAR (αCAR), CD4 (αCD4), or CD19 (αCD19). Black arrowheads point at infiltrated lymphocytes, red arrowheads at histiocytes, and the yellow line indicates B-lymphocyte rich zones.
Fig.Figure 2(a) Synteny for chromosomal regions containing the GPR15-C10ORF99 pair. The pair and their neighbouring genes in vertebrate genome fragments are shown. Deep red shading represents exon 1 of the CDHR1 gene of amniotes. Deep grey color represents exon 1 of the CDHR1 gene of amphibians and fishes. Light red represents exon 1 of the C10ORF99 gene. 3'rd WGD indicated the fishes that underwent a third round of whole-genome duplication. (b) Amino acid sequence alignment of C10ORF99 and CDHR1 signal peptides. (c) Molecular evolution of C10ORF99 from CDHR1. Regions in CDHR1 corresponding to regions in the C10ORF99 gene and nucleotide sequence identities are given. The locations of the protein coding regions of exons and as well as some intron locations are marked, and protein coding regions of exon sequences are bold and capital. Coding means protein coding region in exon.
Figure 8A. Permeability assay using vibratome section showing the cadaverine 555 dye (red), CD31 (green) and CD13 (purple) in the brain vasculature of Glut1iECKO mice with (bottom) or without (top) lactate administration. Note the extravascular accumulation of cadaverine dye in control Glut1iECKO mice, but not in lactate supplemented Glut1iECKO mice. B. Quantification of the CD13-positive pericytes coverage in the brain vasculature of Glut1iECKO mice with (orange) or without (blue) lactate administration. (n=16 from 5 independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.) C. Quantification of the number of cadaverine 555 positive brain parenchyma cells in the brain vasculature of Glut1iECKO mice with (orange) or without (blue) lactate administration. (n=24 from 5 independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.) D. Quantification of CSF lactate level in WT and Glut1iECKO mice with/without lactate supplementation. (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.) E. Kaplan-Meier survival curve of Glut1iECKO mice after tamoxifen injection at P60 with (orange) or without (blue) lactate administration. (n=8)
Figure 2A. Representative low-pass filtered cryo-EM micrographs of MTs decorated with wild-type DCX (WT) or NDC-NDC construct (NN) after 30 sec or 1 h incubation at 37 °C. Additional DCX proteins were added in excess after MT deposition on EM grids to maximise MT decoration. The final ratio of WT or NN to tubulin was 50:5 μM, giving dense protein background. WT co-polymerized exclusively 13-PF MTs, while NN both 13- and 14-PF MTs. Rapid (30 sec) MT polymerization in excess of WT produces MTs in an intermediate GTP/GDP.Pi state, whereas long-lived WT-MTs and NN-MTs are all in the GDP state. B. Isosurface +end and side views of cryo-EM reconstructions of MTs decorated with different DC domains: α-tubulin, light violet or light grey; β-tubulin, dark violet or dark grey; CDC, yellow; NDC, blue. C. Close-up views of the DCX binding site focusing on loop 3 density, which differentiates DC domains. Refined atomic models in predominantly backbone representation are fitted to their respective density maps (wireframe). Selected residues are labelled with single letter codes and selected side chains are shown as sticks. Nucleotide in tubulin subunit designated as β1 is shown as sticks in the top left corner of each panel. Colours as in (B), or oxygen atom, red; nitrogen atom, navy blue. D. Cartoon illustration of the modes of WT or NN binding to MT lattice, based on cryo-EM results. WT decorates the GTP/GDP.Pi lattice predominantly via CDC ('C') or via both DC domains ('N-C') and the GDP lattice predominantly via NDC ('N') or via both DC domains ('N-C'). NN can decorate the GDP lattice via its N-terminal NDC ('N'), C-terminal NDC ('C') or both ('N-C'). Modes 'N' and 'C' are compatible with MT cross-linking (bundling) by NN. E-G. Assessment of the model-map fit. Upper panels show single DC domains bound to four tubulin subunits: t, MT polymerization time; density maps, semi-transparent isosurface; models, backbone. Lower panels show close-up views of the MT-facing side of the DC domains (backbone + side chains shown as sticks). The main differentiating features between NDC and CDC are highlighted with dashed lines. NDC density in the NN reconstruction (G) results from averaging of two NDCs with varying N-terminal region, causing less defined density for that region (N-term scrambling, shown without side chains). Colouring as in (C).
Figure 4Representative Western blots generated using extracts from cells of expressing oncogenic HRAS and controls treated with ML-SI1 as indicated. The primary antibodies used are indicated on the right. Bar graph showing quantification of the Western blots shown in (A). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test. Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations. Primary antibodies used are indicated on the right. Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations. Primary antibodies used are indicated on the right. Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations. Bar graph showing quantification of the Western blots in (C). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test. Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations. Bar graph showing quantification of the Western blots in (D). All values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.
Figure 6(A) Western blot analysis of PARP1 in chromatin. Two Ewing Sarcoma cells (CHP100 and A4573) were treated with MMS (0.02%, 1 hour) with or without release (1 hour) from MMS (N.T: non treat, T: treat, R: release sample). (B) U2OS and two Ewing Sarcoma cells were treated MMS (0.02%, 30 minutes) and subjected to immunofluorescence by PARP1 antibody after chromatin bound fraction. Scale bar indicates 20 μm (left). Red line indicates mean and more than 300 cells were analyzed. Significance determined by Two-way ANOVA, ***P < 0.001. (C) Human Ewing Sarcoma histology samples were analyzed by immuno-histochemistry using PAR antibody. Scale bar represents 100 mm. (D) Model of EWS role in genomic integrity.
Figure 3 (A) Sox9, Slc1a3, Mbp and Rbfox3 expression in astrocytes that have been arranged according to a pseudo-temporal order. Dashed lines denote threshold levels RPKM=1 (B) Sox9, Sox10, Mbp and Rbfox3 expression in oligodendrocytes that have been arranged according to a pseudo-temporal order. Dashed lines denote threshold levels RPKM=1 (C) 4000 randomly selected genes expressed in astrocytes (threshold RPKM >2) were clustered into four groups (A1-A4) according to their expression patterns along the pseudo-time axis (D) 4000 randomly selected genes expressed in oligodendrocytes (threshold RPKM >2) were clustered into four groups (O1-O4) according to their expression patterns along the pseudo-time axis (E) Enrichment of astrocyte specific genes (from scRNA-seq) and genes bound by SOX3 and SOX9 in GPCs in the four different astrocyte gene clusters (A1-A4), shown in Fig. 3C. Three-star significance equals p-value e-13 (prop.test, R) based on the size of the gene groups (A1=740, A2=1019, A3=882, A4=1236) (F) Enrichment of oligodendrocyte specific genes (from scRNA-seq) and genes bound by SOX3 and SOX9 in the four different oligodendrocyte gene clusters (O1-O4), shown in Fig. 3D. Three-star significance equal p-value e-7 for cluster O3 and e-16 for cluster O4 (prop.test, R) based on the size of the gene groups (O1=1371, O2=1451, O3=839, O4=141)
Figure 3Schematic representation of the assumed relationships between proliferating fibroblasts (PF), quiescent fibroblasts (QF) and extracellular matrix (ECM) during dermal maturation. PF self-replicate with rate κ1. PF and QF can switch among them with rates κ2 and κ-2 respectively. QF deposit ECM at rate κ3. ECM promotes the switch from PF to QF at rate κ4. (B) System of ordinary differential equations (ODE) describing the process of dermal maturation. Because the system is dynamic, all entities can decay over time (κ6 is the degradation rate of QF, κ7 is the degradation rate of ECM, κ5 is the degradation rates of PF and is incorporated into β as β = κ1GF - κ2 - κ5 where GF stands for the Growth Factors we assume exist). Resulting simulation of the system of equations
Figure 5The Kaplan-Meier curve demonstrates increased survival of cct3A RNAi animals when treated with DTT under starved conditions. Percentages indicate the number of survivals at the indicated time points. Three asterisks indicate p < 0.001 with log-rank (Mantel-Cox) test. Images are representative surviving animals for the different conditions at the displayed time point of regeneration. At the bottom are the number of planarians with the phenotype shown. The rest of planarians are dead at the displayed time point.