EMBO/sd-character-level-ner huggingface dataset:
Figure 6(A) Haemotoxylin and Eosin (H&E) staining of toes from forepaws from WT and RDEB mice treated with daily injections of 0.1, 1.0 mg/kg Ang-(1-7) or PBS for seven weeks. Inflammatory cells are elevated in the toes from the PBS and the two Ang-(1-7) treated RDEB mice compared to the WT counterpart. Scale bar = 100 μm. Data information scale bar = 100 μm. (B-D) Flow cytometry analysis of the number of CD45 positive cells (B), percentage of neutrophils (C) and of inflammatory macrophages (CD38+Egr2-) and tissue-repair macrophages (CD38-Egr2+) (D) in forepaw skin of RDEB mice treated as in A. Data information: individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant and shown; n = 5-14 biological replicates. (E) The mean fluorescence intensity (MFI) of MHC II staining on neutrophils and macrophages, inflammatory macrophage (CD38+Egr2-) and tissue-repair macrophages (CD38-Egr2+) from mice as in B-D. Data information: , individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant and shown; n = 5-14 biological replicates. (F) Staining for IgG (red) in forepaws, nuclei counterstained with DAPI. (G) Quantification of fluorescence staining intensity of samples as in F. Data information: scale bar = 100 μm. , individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant and shown; n = 5-14 biological replicates.
Figure 4A, HCT116 were treated as indicated for 24 hrs, subjected to propidium iodide staining and analyzed by fluorescence-activated cell sorting (FACS). Data are representative of at least three independent experiments. B, Colon adenocarcinoma cell lines HCT116 (circles), LoVo (squares), LS174 (triangles), or RKO (inversed triangles) were treated with the vehicle alone (-), 1 µM MPA or 10 µM MPA for 24 hrs and subjected to propidium iodide staining and FACS analysis. Data are shown as the fold induction of the cell number in G0/G1 phases (blue), S phase (red), and G2/M phases upon treatment with 1 µM MPA (dark) or 10 µM MPA (clear) over vehicle treated cells. C, IdU track length distribution, and distribution of long fork / short fork IdU track length ratios (insert). IdU track length of at least 300 fibers was measured for each condition. For fork symmetry analyses, IdU track length was measure in at least 38 bidirectional forks (see methods). ***p<0.001, ****p<0.0001 by Kruskal-Wallis test of the means. D, Representative images from (C). E, HCT116 cells, pre-incubated with 10 µM BrdU for 48 hrs were treated with Vehicle (-), 10µM MPA for 6 or 24 hrs, or 20 µM Hydroxyurea for 4 hrs before cell permeabilization and fixation (see Method). Cells were immunostained for native BrDU (green), or RPA (red). DNA was counterstained with DAPI (blue). One representative z-confocal stack is shown per condition. Scale bars correspond to 5 μm. F, HCT116 cells treated with 10 µM MPA for increasing time were analyzed by Western blots for the indicated proteins. α-tubulin was used as a loading control.
Figure 1Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in leptotene Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in zygotene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in , early pachytene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in mid pachytene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in diplotene Arrows in (I-L) indicate the position of some chromocenters. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. DAPI staining of the chromatin (blue) is shown for some spermatocytes. Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in diakinesis Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. O, P Double immunolabeling of PDS5A (green) and kinetochores revealed with an ACA serum (red) in a metaphase I spermatocyte. Data information: DAPI staining of the chromatin (blue) is shown for some spermatocytes. Q, R Double immunolabeling of PDS5A (green) and REC8 (pseudocolored in purple) in a metaphase I spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. DAPI staining of the chromatin (blue) is shown for some spermatocytes. S, T Double immunolabeling of PDS5A (green) and kinetochores (ACA, red) in a metaphase II spermatocyte. Data information: DAPI staining of the chromatin (blue) is shown for some spermatocytes.
Normal phenotypes of the nbr1 mutant plants in resistance to Botrytis.Figure 9(A) Responses of Col-0 wild type (WT), atg5, atg7 and nbr1 plants to Botrytis. Wild-type and mutant plants were inoculated by spraying with spore suspension at a density of 2.5×105 spores ml−1, and kept at high humidity. Pictures of representative plants were taken at 4 and 5 days post inoculation (dpi). (B) Quantitative real-time PCR analysis of the B. cinerea ActA (BcActA) transcript levels in infected Arabidopsis plants at 5 dpi. The experiments were repeated twice with similar results.
Figure 2(A) Representative EPSP and mEPSP physiological traces from larval NMJs at Muscle 6. Recordings were taken from WT control larvae (Elav-Gal4>UAS-RFP), hiw mutants driving a control transgene (hiwND8;Elav-Gal4 > UAS-RFP), and hiw mutants expressing RNAis to knockdown either dNmnat (hiwND8;Elav-Gal4 > UAS-dNmnat-RNAi [#29402] ) or Wnd (hiwND8;Elav-Gal4 > UAS-Wnd-RNAi [#25396]). (B) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [WT n = 10 cells, hiw n = 10 cells, hiw > Wnd-RNAi n= 10 cells, hiw > dNmnat-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=39, F=14.02, p<0.0001. WT vs hiw p = 0.0012 (**), WT vs hiw > Wnd-RNAi p < 0.0001 (****), WT vs hiw > dNmnat-RNAi p = 0.9880 (NS)]. (C) Quantification of quantal content (±SEM). [WT n = 9 cells, hiw n = 9 cells, hiw > Wnd-RNAi n= 10 cells, hiw > dNmnat-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F= 2.508, p<0.0001. WT vs hiw p = 0.0361 (*), WT vs hiw > Wnd-RNAi p = 0.0001 (***), WT vs hiw > dNmnat-RNAi p = 0.9889 (NS)]. (D) Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in panels A-C. NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red). (E) Quantification of the mean (±SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [WT n = 23, hiw n = 21 , hiw > Wnd-RNAi n= 16, hiw > dNmnat-RNAi n=19. 1-way ANOVA w/Tukey's multiple comparisons, DF=78, F=169.1, p < 0.0001. WT vs hiw p < 0.0001 (****), WT vs hiw > Wnd-RNAi p = 0.9988 (NS), WT vs hiw > dNmnat-RNAi p < 0.0001 (****), hiw vs hiw > dNmnat-RNAi p = 0.9695 (NS)].
Structure‐based analysis of Gli1/DNA complexRepresentative Gli1ZF/DNA structure extrapolated from MD trajectories.Figure 1A Representative Gli1ZF/DNA structure extrapolated from MD trajectories. Gli1ZF is shown as blue cartoon, residues involved in binding to DNA (based on single point mutation study) are shown as magenta sticks, and Zn ions as gray spheres. B Effect of Gli1ZF mutants on the binding affinity to DNA as predicted by in silico alanine scanning. The ∆∆G was calculated along MD trajectories as the difference between the ∆G of each Gli1ZF alanine mutant and Gli1ZF‐WT. Results are shown as ∆∆G values in kcal/mol calculated by means of the MM‐PBSA methods ± SEM. C Effect of Gli1ZF mutants on Gli1‐dependent transcriptional activation. Luciferase assay was performed in HEK293T cells transfected with 12XGliBS‐Luc (GliBS, Gli binding site), pRL‐TK Renilla (normalization control), Flag‐Gli1 WT or the indicated Flag‐Gli1 mutants. Data show the mean ± SD of three independent experiments. *P 0.01; **P 0.05 versus Gli1 WT. Western blot analysis of Flag‐Gli1 WT or the indicated Flag‐Gli1 mutant expression levels (bottom panel). D Specific H‐bond interactions between K350 of Gli1ZF and Gli1 binding site consensus, as predicted by MD. E Gli1/DNA binding. Double‐stranded oligonucleotide containing the canonical GliBS sequence (5′-TTGCCTACCTGGGTGGTCTCTCCACTT-3′) or mutated GliBS sequence used as control (5′-TTGCCTACCTCCCACTTCTCTCCACTT-3′) was used as probe (P) in EMSA experiments. The assay was performed using recombinant GST‐Gli1ZF‐WT (Gli1 zinc finger fragment: aa 242-424), GST‐Gli1ZF‐K350A and GST‐Gli1ZF‐K340A. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF mutants bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding (as described in ). *P 0.05 versus Gli1 WT.
Figure 4Scheme of the identification of Myoparr-interacting proteins using RiboTrap and differential proteomics analysis. Following immunoprecipitation, the interaction between Myoparr and endogenous Ddx17 was confirmed by immunoblotting using a Ddx17-specific antibody. Two different Ddx17 isoforms (p72 and p82) were observed. Determination of the Ddx17-binding region of Myoparr by RNA pull-down analyses. Schematic diagram of full-length or truncated Myoparr used for RNA pull-down (top). In vitro transcribed/translated Ddx17 protein was pulled down by indicated Myoparr and then detected by western blot using a Ddx17 antibody (bottom). Schematic diagram of the constructs used for luciferase assays. The details of all constructs are described in Materials and Methods. p(A) indicates poly(A) site. Relative luciferase activities of indicated constructs in differentiating C2C12 cells. Relative luciferase activities of indicated constructs in differentiating C2C12 cells transfected with control or Myoparr shRNA (Myoparr KD).
+-sensitivity of Nxph1-LNS2 interactions.Figure 4Representative fluorescence polarization binding curves are shown for LNS2SS2- and LNS2SS2A+ interacting with Nxph1 with or without Ca2+ added. Comparison of binding affinities of Nxph1 for Nrxn1 LNS2SS2- and Nrxn1 LNS2SS2A+ with or without Ca2+ added; replicate numbers are indicated in bars. Error bars represent SEM, and significance values were calculated using Welch's t-test. ns, not significant.
Figure 2(A) Immunoblotting analysis of ESM1 in supernatant (S), nucleus (N), and cytoplasm (C) was performed. Ponceau S, Lamin A/C, and α-tubulin were used as loading control in supernatant, nucleus, and cytoplasm, respectively. P: parental cells, M: metastatic cells. (B) Left, representative images of cells in the transwell invasion assay. Right, quantification of cells that invaded through the matrigel-coated membrane following treatment with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody. Scale bar: 100 μm. (C) Schematic diagram of ESM1 expression constructs. (D) Elicitation of ESM1 overexpression using HA-tag antibody results in detectable ESM1 expression in the nucleus and cytoplasm of 22Rv1-P cells. Lamin A/C and α-tubulin were used as loading control in the nucleus and cytoplasm, respectively. (E) Immunoblotting of ESM1 expression with different expressing patterns in the supernatant (S) of 22Rv1-P cells. (F) Left, representative images of 22Rv1-P cells in the transwell invasion assay. Right, statistic data of invasive ability of ESM1 expressing cells. Scale bar: 100 μm. (G) Left, representative bioluminescence images of lung metastasis in mice after tail vein injection of 22Rv1-Luc cells with different ESM1 expression patterns. Scale bar: 0.5 cm. Right, quantification of the lung bioluminescence by photons/ seconds.
Fig.Figure 1Representative images of primary neurons isolated from superior cervical ganglia (SCG) and sensory trigeminal ganglia (TG) of postnatal (P6) and adult (P28) mice stained for PML and the neuronal marker BIII-tubulin. SCG and TG neurons isolated from P6 and P28 mice were treated with interferon (IFN) α (600 IU/ml) for 18h and stained for PML and BIII-tubulin. Quantification of detectable PML puncta in P6 and P28 neurons following 18h treatment with IFNα (150 IU/ml, 600 IU/ml), IFNγ (150 IU/ml, 500 IU/ml) and IFNλ2 (100 ng/ml, 500 ng/ml). Data information: Data represent the mean ± SEM. n=60 cells from 3 biological replicates. Statistical comparisons were made using a one-way ANOVA with a Tukey's multiple comparison (ns not significant, **** P<0.0001). Scale bar, 20μm.
EB2 S223 phosphorylation is lost in CDD patient-derived neurons.Figure 7 A, B Western blots for total EB2, pS223 and tubulin using protein lysates obtained from neurons derived from three patients with CDD and their related control. 1 = p.D135_F154del, male; 2 = p.R59X, male; 3 = p.R59X, female. Quantification of EB2 phosphorylation is normalized for total protein level. Student's t-test: n = 3 patients/controls. * p < 0.05, error bars are SEM  C Representative images of human iPSC-derived neurons co-stained with the neuronal dendrite marker MAP2, EB2 and EB2 pS223. EB2 phosphorylation is present in dendrites of control neurons but not in CDD neurons. Enlarged images of the boxed areas are shown on the right panel. Scale bar is 20 μm
Figure 6A. YAP S128D reconstituted cells show growth advantage under hyperosmotic conditions. YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D were cultured in the absence (left panel) or presence of 0.2M sorbitol (right panel) for the indicated amount of time. Cell numbers were counted and normalized to day 0. Data are presented as mean ± SEM, n = 3. B. YAP S128D reconstituted cells have lower cell death in a hyperosmotic environment. Cell cycle analyses of YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D after 0.2 M sorbitol treatment were determined using flow cytometry. Propidium iodine (PI) was used for DNA staining (Fig EV5). Quantification of sub-G1 cells is shown. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3). C. YAP S128D reconstituted cells show reduced apoptosis under hyperosmotic conditions. Annexin V analyses of YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D after 0.2 M sorbitol treatment were determined using flow cytometry. PE-Annexin V and 7-AAD stained for phospholipid phosphatidylserine (PS) and DNA, respectively. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3). D. A proposed model for osmotic stress regulation of YAP via NLK Kinase. See Discussion for details.
Figure 2IFM images of KIF13A-YFP and mCherry-Rab22AWT/Q64L/S19N cotransfected HeLa cells. Arrowheads and arrows point to the KIF13A-positive tubular REs and E/SEs respectively. The colocalization coefficient (r, in mean±SEM) between two markers was indicated separately. Scale bars: 10 μm Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 2A. n=3. nc: total number of cells. nt: total number of tubules Subcellular fractionation of HeLa cells to probe the localization of Rab22A (red box) with respect to other organelle specific proteins. *, non-specific bands.
Figure 5Left: Data generated as described above in Figure 2 was used to generate a heatmap of homologous recombination (HR) gene expression after the indicated treatment regimens. Middle: Selected GSEA MSigDB Oncogenic Signature pathways are shown. Right: Data generated as described above in Figure 2 was compared to a previously described HR deficiency transcriptional profile (Peng et al., Nature Communications, 2014). This profile was derived by independently silencing either BRCA1, RAD51, or BRIP1, followed by transcriptional analyses. The union of these three data sets was used to generate the signature. Cut-offs for comparison were a p value<0.05, and fold change of 1.5. Venn diagrams show the overlapping and non-overlapping genes of both down- (top) and up-regulated (bottom) genes in the previously-defined HR deficiency signature, and the PARPi-responsive transcriptome Left: C4-2 cells were treated as depicted in Figure 2. Data are depicted as mean +/- standard deviation of three independent biological experiments. Statistical significance was determined by two-tailed Student"s t test where *=p<0.05, **=p<0.01. BRCA2, p=0.0046; RAD51, p=0.0151; XRCC3, p=0.0341; TOP3A, p=0.04988. Right: C4-2 cells were treated as depicted in Figure 2 and immunoblotted with the indicated antisera. Quantifications shown below each Athymic nude mice were injected with C4-2 cell mixed with matrigel. Once tumors became 100mm3, mice were treated with either vehicle control or veliparib. 72 hours later, tumors were harvested, RNA was isolated and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples, +/- standard deviation of three independent xenograft tumors Prostatectomy tissue (n=6) was cultured as previously described, and treated with either vehicle control or veliparib for six days. RNA was then harvested from the tissues and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples. Each individual tissue is depicted by a separate bar color. Statistical analyses were performed by Wilcoxon signed rank test C4-2 cells were treated as depicted in Figure 2. ChIP was performed using the indicated antisera, and the subsequent DNA was isolated and used in qPCR reaction using primers designed to amplify the indicated genomic loci: BRCA2 enhancer, RAD51 promoter, or TOP3A promoter C4-2 cells treated with 2.5uM veliparib (Vel.) or vehicle control (Veh.) for 24 hours. Cells were then harvested, lysed, and differentially centrifuged as described in the material and methods section, resulting in a soluble fraction (Sol.) (GAPDH serves as control) or a chromatin-tethered fraction (Teth.) (histone H4 serves as control). Immunoblots were performed for the indicated proteins
Figure 2A. PPP1R13L mRNA expression level is lower in VI10 PDFs (set as 1) than in CFs. B. and C. Western blots (WBs) analyses demonstrated the absence of iASPP in VI10, VI4 and VII1 PDFs. D. WB analysis demonstrated that PPP1R13L-shRNA downregulated the putative iASPP band.
Figure 2C57BL/6J male mice fed on a chow diet drank water with or without asparagine at a concentration of 1.5g L-1 for 3 weeks. Mice were acutely exposed to 4˚C for 6 h to measure rectal temperature or intraperitoneally (i.p.) injected CL316,243 (1mg kg-1 body weight) for one time for other assays. Mice serum amino acid concentration after 3-weeking asparagine feeding. n=5 mice each group. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test. Asparagine content in BAT of mice in (B). n=5 mice each group. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test. Rectal temperature was measured when mice in (A) was challenged to 4˚C. n=5 mice each group. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test. E,F. Serum insulin (E) and triglyceride (F) concentration was assayed in mice 24 h after one dose of CL316243 injection. n=5 mice each group. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test. UCP1 and PPARγ protein levels in BAT and inguinal WAT (iWAT) from mice in (E). Representative pictures of BAT and inguinal WAT (iWAT) hematoxylin and eosin staining from mice in (E). Scale bar: 20μm for BAT, 50μm for iWAT. I,J. Mice supplemented with asparagine in drinking water for 3 weeks were subjected to the indirect calorimetry analysis at both basal (I) and CL316,243-stimulated (J) states. n=5 mice each group. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.
The origin of N-terminal proteoforms.Figure 1Figure 1 - The origin of N-terminal proteoforms. A. 2,578 proteoforms were identified including 2,135 dbTIS and 443 aTIS. For the majority of proteins we found a unique translation initiation site (grey). Additionally, 335 N-terminal peptides pointed to proteins with multiple N-termini created by alternative translation initiation (blue), whereas 81 N-termini were generated by alternative iMet processing (green). B-C. Proteomics-derived translation initiation events were verified at the transcript level using Ribo-Seq data obtained in Jurkat cells and HCT 116 cells (data unpublished). Although in this study, the degree of in vivo Nt-acetylation could not be directly determined, all retained N-termini were compliant with the rules of N-terminal processing from previous experiments (Van Damme et al, 2014). In the case N-termini were found to be in vivo Nt-acetylated (previous MS/MS-based evidence (Van Damme et al, 2014)) this information is indicated. dbTIS and aTIS peptides were further mapped to protein N-termini annotated in Swiss-Prot Isoform, UniProt TrEMBL and Ensembl databases and neo-N-termini (proteolytic products) of the knowledgebase TopFind 3.0. The number of matching N-termini is indicated in dark grey. N-terminal peptides supported by at least one source of metadata pointing to translation (Nt-acetylation, Ribo-Seq, Swiss-Prot Isoforms, TrEMBL or Ensembl) were classified as highly confident TIS (indicated in green). D. Using experimental and metadata we assigned the most likely origin of aTIS peptides and confirmed that alternative translation initiation within the same transcript (leaky scanning) contributes to the majority of alternative TIS identified.
Model for regulation of autophagy through mAtg9, p38IP, and p38 MAPK.Figure 8Model for regulation of autophagy through mAtg9, p38IP, and p38 MAPK. In full medium, mAtg9 traffics between the TGN and endosomes. Autophagy is inhibited by phosphorylated p38α. p38IP is found in the pool of mAtg9 in peripheral endosomes, and in complex with p38α. It should be noted that the two pools of p38α-p38IP are identical and shown separately for clarity. In starvation, p38α is dephosphorylated and binds p38IP with a lower affinity, and can no longer inhibit autophagy (shown as a dashed line). The pool of p38IP released from p38α would facilitate mAtg9 trafficking and autophagy.
The ribose-salvage pathway is essential for yeast's survival in starvation.Figure 7(A) Level of hallmark metabolites and energy charge in wild type, phm8 and pnp1/urh1 strains in starvation. The x axis represents hours after starvation, and the logarithmic y axis represents energy charge ([ATP]+0.5[ADP]/([ATP]+[ADP]+[AMP]) or absolute intracellular concentration (mean±range of N=2 biological replicates). (B and C) Growth and viability of wild type, phm8 and pnp1/urh1 strains in starvation. (D) Growth of wild type, phm8 and pnp1/urh1 strains in the switch from glucose to glycerol+ethanol. The x axis represents hours after starvation and the logarithmic y axis represents optical density (A600) in (B and D) and percentage of live cells calculated by dividing the number of colonies formed after starvation by the number of colonies formed before starvation in (C) (mean±range of N=2 biological replicates).