EMBO/sd-character-level-ner huggingface dataset:
Figure 1A Pluripotent stem cells (PSCs) with one of two distinct exonic loss-of-function mutations (Q83X nonsense or K82 frameshift (K82fs)) at the MECP2 locus were generated and differentiated into neurons via a neural progenitor cell (NPC) intermediate. B Immunofluorescent staining confirmed the absence of MeCP2 in MECP2-KO PSCs, NPCs, and neurons. Scale bar = 10 µm. C RT-qPCR array revealed differential gene expression between control and MECP2-KO 28-day neurons. The P values are calculated based on a Student's t-test of the replicate 2-ΔΔCt values for each gene in the control group and KO groups (WT83/Q83X cell lines were used; N = 3 clones from each genotype). D Overtime quantitative gene expression profile of many of the involved genes relevant to synaptic function (days 14 and 28 post differentiation; normalized by control NPC expression). E Single-cell RT-qPCR analysis distinguished populations of control and KO 28-day neurons. Principal Component Analysis (PCA) of 441 cells projected onto the first two components. Overlaid populations of WT83/Q83X and WT82/K82fs neurons are shown. F Violin plots of selected genes showing the comparison between control and MECP2-KO neurons from the single-cell analyses (log2(expression) values) that overlapped with the results obtained via RT-PCR array. G Volcano plot illustrates differences in expression patterns of target genes of neurons from the qPCR single-cell analyses. The dotted lines represent differentially expressed genes between the groups at P < 0.05 (unpaired Student's t-test). H Left: Schematic of laminar cortical markers. Right: Top: Quantitation of laminar cell markers based on the single-cell RT-qPCR analysis (two-way analysis of variance (ANOVA), N = 3 replicates per genotype, F3,24 = 3.31, *P = 0.04, with difference in the proportion of cortical layers V and VI, 95% confidence interval (CI) [8.31, 42.41]); Bottom: altered proportions of neurotransmitter markers (two-way ANOVA, N = 3 replicates per genotype, F5,24 = 27.33, ***P < 0.0001, with differences in the proportions of glutamatergic, 95% CI [22.33, 33.60], and cholinergic, 95% CI [0.166, 11.43], neurons). I Gene ontology (GO) analysis of targeted genes relevant to synaptic function displaying the differences between MECP2-KO and control neurons. For the full list of markers used see Dataset EV1.
a) HeLa cell lysates were immunoprecipitated with Atg16L1 antibody (Atg16L1) or no antibody (No Ab) in control, and subjected to SDS–PAGE; proteins were stained with SimplyBlue safe stain (Invitrogen) in accordance with the manufacturer's protocol.figf1(a) HeLa cell lysates were immunoprecipitated with Atg16L1 antibody (Atg16L1) or no antibody (No Ab) in control, and subjected to SDS-PAGE; proteins were stained with SimplyBlue safe stain (Invitrogen) in accordance with the manufacturer's protocol. Bands indicated in the box were cut out and digested with trypsin, and identified by MALDI-TOF MS. HC, heavy chain. (b) HeLa cells transfected for 24 h with control vector, or wild-type or different deletion mutants (residues 2-77, 2-275 and 232-607) of Flag-Atg16L1 were immunoprecipitated with anti-Flag antibody (Atg16L1) and immunoblotted with anti-clathrin, anti-Flag and anti-Atg5 antibodies. Clathrin interacts with the N terminus of Atg16L1, in a similar manner to Atg12-Atg5. Total lysates were run alongside as controls for protein input. FL, full-length. (c) HeLa cell lysates were immunoprecipitated (IP) with Atg16L1 antibody (+Ab) or no antibody (no Ab) and immunoblotted with anti-clathrin, anti-AP2 and anti-Atg16L1 antibodies. Total cell lysates (TL) were also blotted for input controls. Endogenous Atg16L1 interacts with clathrin heavy chain and AP2. (d) HeLa cells transfected for 24 h with Flag-Atg16L1 and increasing concentrations of Atg5 were immunoprecipitated (IP) with anti-Flag antibody (Atg16L1) and immunoblotted with anti-clathrin, anti-Flag and anti-Atg5 antibodies. Atg5 overexpression neither interferes with nor increases clathrin-Atg16L1 binding. Uncropped images of blots are shown in Supplementary Information, Fig. S9.
Figure 7qRT-PCR for pri-miR-133b, pri-miR-206, and H19 expression in differentiating C2C12 cells transfected with indicated siRNAs. n = 3, mean ± SD. *p < 0.05. **p < 0.01. ChIP-qPCR detection of Pol II occupancy at the indicated promoters in Myoparr-depleted C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. Myoparr KD decreased Pol II occupancy at the miR-133b promoter with a marginal trend toward significance (p = 0.052). ChIP-qPCR detection of Ddx17 (C) occupancies at the indicated promoters in Myoparr-depleted C2C12 cells. n = 3, mean ± SD. *p < 0.05. **p < 0.01. ChIP-qPCR detection of PCAF (D) occupancies at the indicated promoters in Myoparr-depleted C2C12 cells. n = 3, mean ± SD. *p < 0.05. **p < 0.01. qRT-PCR showing decreased expression of miR-133b and miR-206 in both Myoparr- and Ddx17-depleted C2C12 cells. n = 3, mean ± SD. *p < 0.05. **p < 0.01. Western blots showing increased ERK1/2 activity (pERK1/2) and Cdc6 expression in differentiating C2C12 cells 48 h after Myoparr and Ddx17 KD. Tubulin expression served as an internal control. Increased Pola1 expression detected by qRT-PCR. n = 3, mean ± SD. **p < 0.01.
Interaction of Cx43 with miR-133b in vitro is increased in the presence of hnRNPA2B1 and hnRNPQ.Figure 6Representative maximum projection images of microscopy-based miRNA-protein interaction using beads coated with GST-tagged NT/CT-Cx43, hnRNPA2B1 or hnRNPQ, as indicated, following the addition of Cy3-labelled miR-133b or miR-509-3p. Graph depicts quantification of total fluorescence/bead, as a measure of protein-miRNAs binding. Scale bars 50 μm (n=2 biological replicates; average of 23 beads analyzed/condition). Data information: p-values were derived by Kruskal-Wallis (Dunn's post-hoc) test. Bars and error bars indicate mean ± SD in all graphs.
Figure 5Western blot detection of ARS proteins in mascRNA overexpressing (mascRNA) or the scrambled RNA expressing (Scramble) cells. β-Tubulin was used as a loading control.   Quantification of the protein levels in Panel A.   Western blot detection of ARS proteins in HEK cells stably expressing mascRNA (mascRNA), the mutants (Anti-Mut1 and Mut1) or the scrambled RNA (Scramble). β-Tubulin was used as a loading control. Western blot detection of ARS proteins in HEK cells stably expressing mascRNA (mascRNA), the mutants (Anti-Mut1 and Mut1) or the scrambled RNA (Scramble). β-Tubulin was used as a loading control. Quantification of the protein levels in Panel C. qRT-PCR analysis of MALAT1 RNA levels in MALAT1 knockdown and the control cells. Northern blot analysis of mascRNA in MALAT1 knockdown and the control cells. 5S rRNA was used as an internal control. Western blot detection of ARS proteins in MALAT1 knockdown and the control cells.   Quantification of the protein levels in Panel G.   qRT-PCR analysis of GARS and QARS mRNAs in mascRNA overexpressing (mascRNA) or the scrambled RNA expressing (Scramble) cells. Western blot detection of ARS proteins in mascRNA overexpressing (mascRNA) or the scrambled RNA expressing (Scramble) cells, before and after emetine and cycloheximide treatment. β-Tubulin was used as a loading control.   Quantification of the relative QARS protein levels in Panel J.   Quantification of the relative GARS protein levels in Panel J.
SMCHD1 loss does not affect recombination frequency at telomeres in TRF2 depleted cells.Figure 3A Western blot detection of ATM pS1981, SMCHD, TRF2, γH2AX, and hnRNPA1 in WT or SMCHD1 KO Hela cells transfected with shTRF2 and EV control. B Representative images for CO-FISH on metaphase spreads in wild type (WT) Hela and U2OS cells. The leading strand telomeres were labeled by PNA-FISH with a (TTAGGG)3-Alexa488 probe (green), the lagging strand with a (CCCTAA)3-Cy5 probe (pink), and the DNA was stained with DAPI. Examples of telomere sister chromatid exchange (t-SCEs) events are marked with white arrows. Scale bar: 5µm C Quantification of chromosome ends with t-SCEs detected by CO-FISH. Data represent means of 2 independent experiments± SD (>700 telomeres/condition/experiment) with individual average values indicated.
Figure 5(A) Proteins and metabolites differentially accumulating in response to the Tyr-Asp feeding were delineated using two-way ANOVA embedded in MeV software Howe et al., 2011(), followed by False Discovery Rate (FDR) correction. Heat map representation of the respectively, 212 and three differential (two-way ANOVA; FDR corrected p≤0.05) proteins and metabolites across five time-points. Data is presented as log2 fold change between control and treatment. Red indicates down- whilst blue up-regulation. 1: log2 fold change (FC) 15 min; 2: log2 FC 30 min; 3: log2 FC 1 h; 4: log2 FC 6 h; 5: log2 FC 24 h; M: median log2 FC of all time points. (B) Median of the log2 fold change between control and treatment calculated from the five time-points was used for MapMan visualization Thimm et al, 2004() of the differential proteins (squares) and metabolites (circles). Blue indicates up- whilst red down-regulation. MapMan automatically assigned Bincodes and proteins were classified into different cellular functions/processes. Bind.: binding; CHO: carbohydrates; Misc.: miscellaneous; Modif.: modification; N: nitrogen; OPP: oxidative pentose phosphate pathway; Reg.: regulation; S: sulfur; Targ.: targeting; TCA: tricarboxylic acid cycle. (C) Venn diagram comparison of putative Tyr-Asp interactors identified in the AP experiments using different sources of starting material. (D) Venn diagram comparison of putative Tyr-Asp interactors identified in the AP, TPP and PROMIS experiments. (E) Cytoscape Shannon et al., 2003() visualization of the Tyr-Asp interactome. Nodes represent Tyr-Asp interactors, edges were imported from the STRING database Szklarczyk et al., 2017() using experimental, database, and literature evidence (score set at 0.4). Functionality was assigned based on the Uniprot Apweiler et al, 2004(). Disconnected nodes were removed from the network.
Fig.Figure 3(A) ATZ glycosylation mutants and their electrophoretic mobility. N is the acceptor site asparagine; Q is for the mutation to glutamine that prevents glycosylation. The E342K mutation of ATZNNN is in blue. (B-I) Confocal laser scanning microscopy analyses (as in Fig. 2) in WT MEF treated with BafA1. (J, K) (J) Quantification of ATZxxx-positive endolysosomes and (K) of ATZxxx polymers-positive endolysosomes (mean, n= 18, 30, 24, 14, 27, 22, 27, 18 cells for HA stain and n= 12, 14, 14, 11, 14, 14, 17, 11 cells for 2C1 stain of panels B-I, respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001.
Fig.Figure 3 A. Upf1 fragments tested: FL is for the full-length protein; HD-Cter for the region containing the helicase domain and the C-terminal part of Upf1 (208-971); CH for the N-terminal domain (2-208) that contains the N-terminal unstructured region and the Cysteine-Histidine rich domain; CH-HD for a version a full-length Upf1 lacking the C-terminal region 854-971; and HD for the helicase domain of Upf1 alone (not to scale). N-terminal TAP-tagged Upf1 versions were expressed from a single copy vector under an inducible tetO7 promoter  B-D. Results of purifications using Upf1-FL (B), Upf1-CH (C as bait with the x-axis showing the average enrichment value (log2 scale) for Upf1-decapping and Upf1-23 components  B-D. Results of purifications usin Upf1-Cter (D) as bait with the x-axis showing the average enrichment value (log2 scale) for Upf1-decapping and Upf1-23 components. The colour of the bars illustrates the treatment used during the purification, black without RNase, and orange with an RNase treatment. Error bars represent SD. White dots correspond to enrichment values obtained in individual experiments  E. Comparison of the enrichment of Upf1, Nmd4 and Ebs1 in the purification of the Upf1 fragments. The Upf1 C-terminal region (854-971) affected the association with Nmd4 or Ebs1 (Student t-test with a one-sided alternative hypothesis). We compared the enrichment of each of Ebs1 and Nmd4 between purifications of Upf1 fragments having this region (Upf1-FL and Upf1-HD-Cter, 6 experiments) and purifications of Upf1 fragments lacking the extension (Upf1-CH-HD and Upf1-HD, 4 experiments). The C-terminal region had no effect on Nmd4 enrichment (p-value ≈ 0.98), whereas there was significantly less associated Ebs1 on this small region (p-value ≈ 0.0013)  F. Western blot of input and eluates of Upf1 domains purification in a Nmd4-HA strain. The band with the # might corresponds to a dimer of Upf1-CH, bands marked with a star correspond to residual signal with the anti-HA antibodies (Nmd4). Fragments in the eluate have a smaller size because the protein A part of the tag was removed by digestion with the TEV protease. G6PDH served as a loading control in the input samples
Figure 1Shown are the two metabolic pathways for GDP-L-fucose synthesis by the salvage and de novo pathways. In the salvage pathway, fucose obtained from diet or lysosomal degradation is converted to GDP-fucose in two steps by L-fucose kinase (FCSK) followed by fucose-1-phosphate guanylyltransferase (FPGT). In the de novo synthesis, which is responsible for most of the GDP-fucose yielded, GDP-fucose is obtained from GDP-D-mannose. The enzymes involved are GDP-D-mannose-4,6-dehydratase (GMDS) and GDP-L-fucose synthase (GFUS). GDP-fucose is then transported to the endoplasmic reticulum (ER) and to the Golgi by transporters SLC35C2 and SLC35C1, respectively. Genes with previously described disease-causing mutations are indicated by their respective MIM numbers.
Figure 2Frequency of WT donor (CD45.1), DKO donor (CD45.2) and recipient (CD45.1/2) cells in the indicated BM (LSK, CMP), splenic (monocytes), liver (Kupffer cells), peritoneal and alveolar macrophage populations of lethally irradiated recipients ≥ 6 weeks after transfer of a 1:1 mixture of lineage-depleted BM cells from WT and DKO mice. P values < 0.05 (paired Student's t-test) between LSK and other populations are shown for frequencies of WT cells. Three mixed BM chimeras analyzed; error bars represent s.d. Representative results of three independent experiments. Representative flow-cytometric analysis of lung (B) macrophages as in A (left). The expression of CD45.1 and CD45.2 on donor and recipient cells is shown next to the indicated gates (right). Representative flow-cytometric analysis of peritoneal (C) macrophages as in A (left). The expression of CD45.1 and CD45.2 on donor and recipient cells is shown next to the indicated gates (right). Frequency of WT donor (CD45.1), DKO donor (CD45.2) and recipient (CD45.1/2) cells among the BM LSK cells, splenic monocytes and alveolar macrophages of the recipients that were irradiated with lung shielding, treated with one dose of busulfan as described in Materials and Methods, transferred with a 1:1 mixture of lineage-depleted BM cells from WT and DKO mice and analyzed ≥ 8 weeks after transfer. Representative flow cytometry results for alveolar macrophages (left) and quantification (right) is shown. Surface phenotype of cells from the digested lungs (right) and quantification of donor contribution to the AM compartment (left) of AM-deficient Csf2rb-/-Csf2rb2-/-recipients (CD45.2) that were subjected to intranasal transfer of a 1:1 mixture of sorted fetal liver monocytes from WT (CD45.1/2) and DKO (CD45.2) E18.5 embryos at days 1 or 2 after birth and analyzed ≥ 7 weeks after the transfer. Cell surface phenotype of lung cells from a Csf2rb-/-Csf2rb2-/- mouse that was not subjected to the transfer is shown for comparison. Frequency of donor- and recipient- derived AMs in WT and DKO recipients transferred at days 1 or 2 after birth intranasally with WT CD45+ cells sorted from neonatal lungs and analyzed ≥ 7 weeks after the transfer. Results for quantification (lower panel) are pooled from 3 independent experiments with a total of 8 WT and 9 DKO recipients; various combinations of CD45.1 and CD45.2 were used to distinguish donor and recipient cells.
Figure 7(A) Decreasing amounts of F-actin were spotted onto a nitrocellulose membrane and binding of indicated DNGR-1 ECD (mouse, long isoform) proteins was tested in PBS or MES pH 6.1 buffers. HRP-conjugated anti-FLAG antibody was used for detection. The signal was quantified by densitometry and quantitation of four independent experiments is shown. (B and C) B3Z-Syk reporter cells expressing indicated mutant or WT DNGR-1 receptors (mouse, long isoform) were incubated with UV-irradiated 293T cells (ratio dead:reporter cells is indicated), or with plate-bound anti-DNGR-1 antibody or medium alone at 37°C overnight. Activation of the NFAT reporter was measured at the end of the incubation period. Data are plotted as mean ± SD of experimental duplicates. One representative of three experiments is shown. (B and C) B3Z-Syk reporter cells expressing indicated mutant or WT DNGR-1 receptors (mouse, long isoform) were incubated with UV-irradiated 293T cells (ratio dead:reporter cells is indicated), or with plate-bound anti-DNGR-1 antibody or medium alone at 37°C overnight. Activation of the NFAT reporter was measured at the end of the incubation period. Data are plotted as mean ± SD of experimental duplicates. One representative of three experiments is shown. (D) Phoenix cells expressing indicated mutant or WT DNGR-1 proteins (mouse, long isoform) were treated with F-actin, F-actin buffer, anti-DNGR-1 antibody or isotype-matched antibody of irrelevant specificity for 45 minutes, fixed in PFA, surface-stained for DNGR-1 and analyzed by flow cytometry. One representative of three experiments is shown.
Figure 3(A) At 36h after transfection of the indicted protease and wtSPINK6 plasmids or blank vector in triplicate, BHK21 cells were inoculated with H7N9/ah at a MOI of 0.5. Cell-free culture media were harvested at 24 hpi for viral titration. Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times. **, P<0.01. Student's t test. (B) At 24h after transfection of HAT or KLK5 plasmid in sextuplicate, A549 cells were inoculated with H1N1/pdm at MOI of 0.25. The infected cells were incubated with recombinant wtSPINK6 or mutSPINK6 protein in triplicate for 24h. Culture media in each well were stored in two aliquots, one was applied to viral titration with conventional plaque assay (black bars), the other was pre-treated with TPCK-trypsin for 1 hour prior to plaque assay (grey bars). Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times. *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.
Figure 1Experimental design for lineage-tracing analysis of secretory cells following genetic depletion of Lats1 and Lats2. Specific time points for tamoxifen induction and tissue analysis are indicated. Representative H&E (top) and immunofluorescent (IF) images (bottom) of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (for secretory cell, white), Acetylated tubulin (Act-Tub, for ciliated cell, green), and DAPI (blue). Scale bar, 100μm. Statistical quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells in (B). Representative IF images of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green), AQP5 (for AT1 cell, white), and DAPI (blue). Scale bar, 100μm. Quantification of Scgb1a1 lineage-labeled tdTomato+AQP5+ AT1 cells in (D). Data are presented as mean ± SEM (n=5 mice for each group). Experimental design for lineage-tracing analysis of secretory cells following genetic depletion of Lats1/2 (double knockout, dKO) and Lats1/2;Yap;Taz (quadruple knockout, qKO). Specific time points for tamoxifen induction and tissue analysis are indicated. Representative H&E (top) and IF images (bottom) of control, dKO, and qKO airways at day 21 post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green), AQP5 (white), and DAPI (blue). Quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells in (G). Data are presented as mean ± SEM (n=3 mice for each genotype).
Figure 6A, B. Co-immunofluorescence of anti-neuronal and anti-regeneration associated proteins in DRG following SCI and treatment with 966 or vehicle. Shown is increased protein expression for regeneration-associated targets in NF200 positive DRG neurons (arrows) after 966 treatment (5 weeks following SCI). Scale bar, 50 μm. (B) Data is expressed as fold change of fluorescence intensity in NF200+ cells, 966 versus vehicle ± s.e.m. N=4-7 biological replicates * p<0.05, ** p<0.01, *** p<0.005 indicate significant difference versus vehicle (Student's t-test).
Figure 3(A, B) WT BMDM were incubated for 1 h without (-) or with (+), the IKKβ inhibitors BI605906 (5 μM), TPCA-1 (5 μM) or PS1145 (10 μM), the TAK1 inhibitor NG25 (2 μM) or the NLRP3 inhibitor MCC950. The cells were then co-stimulated for 30 min without (-) or with (+) 100 ng/ml LPS and/or 4 mM ATP (A) and/or 5 μM nigericin (B). The cell culture medium was removed and the cells lysed. Protein in the culture medium was precipitated (see Methods), dissolved in SDS. and subjected to SDS-PAGE, along with cell lysates. After transfer to PVDF membranes, immunoblotting was performed with antibodies recognizing both full length (FL) and cleaved (CL) gasdermin D (GSDMD), the p20 and p10 fragments of caspase-1 and IL-18. Similar results were obtained in two independent experiments. (C) As in A, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) and control WT cells were used. Similar results were obtained in three independent experiments.
Phenotypic changes in adipocyte-specific QKI-deficient mice.Figure 1(A) The mRNA levels of QKI relative to those of 36B4 in BAT and ingWAT from HFD mice (n = 8) and lean controls (n = 8). WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed a NCD or HFD for 20 weeks. The arrow indicates the time when the high fat diet（HFD）starts, and body weight was monitored weekly. (n = 10, per genotype) Representative pictures of adipocyte-specific QKI-deficient mice under NCD or HFD conditions. Gross appearance of tissues from BAT, eWAT and ingWAT fat pads from WT fl/fl and QKI KO fl/fl:AP2-Cre mice after 20 weeks on the NCD or HFD. H&E staining of BAT, eWAT and ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (Scale bar represents 50 μm) The workflow for administration of AAV-ShQKI or AAV-NC vectors into BAT and ingWAT to suppress QKI expression under HFD conditions. The gross appearance of BAT, eWAT and ingWAT fat pads after administration of AAV-ShQKI or AAV-NC vectors in local adipose (ingWAT and BAT). The increase in relative body weights following HFD feeding. (n = 8, per group) H&E staining of the liver, BAT and ingWAT from AAV-NC or AAV-ShQKI mice. (Scale bar represents 50 μm).
Figure 4A) Immunocompromised mouse leg showing ablated TA (control) and implanted TA (graft) at day 0; inset showing muscle mass removed next to the implanted Mabs loaded constructs shaped to fill the TA lodge. B) Implanted myo-substitute 20 days after grafting. C) Isolated TA from control ablated (control) and ablated/grafted leg (graft), the latter revealing a remarkable enhanced size. D) Graphical representation of the scored TA weight (n=6). The dashed area indicates the range of native TA mass depending primarily on mouse weight and age, statistical significance was analyzed by Anova test (P < 0.05 was considered significant: *=0,0306).
Figure 2Pie chart depicting the various speices of 5'-tsRNAs expressed in the three different states of stem cells tested. Northern blot of candidate tRNAs and tsRNAs tsGlnCTG, tsGlyGCC, tsGluTTC, tsValCAC and tsLysTTT. The black arrows represent the tRNAs and the red arrows mark the tsRNAs. Northern Blot of 5'-tsRNAs; tsGlyGCC, tsGlnCTG, tsGluTTC, tsLysTTT, tsValCAC at various timepoints of RA-induced mESCs differentiation showing dynamic expression of these 5'-tsRNAs. Relative quantification of the tsRNA bands from northern hybridization tested across two biological replicates. The solid lines represent the expression of tsRNAs and the dashed lines represent tRNAs.