EMBO/sd-character-level-ner huggingface dataset:
Figure 5Immunoblot detection of PHD2 and ACTIN protein levels from lysates of normoxic cultures (n=4) or B16-F10 tumors [CntlKD (n=5), BNIP3KD (n=7) and ATG5KD (n=6)]. Densitometric quantifications relative to ACTIN levels are shown below each corresponding band. In vitro data was analyzed using a one-sample T-test against shCntl whereas the in vivo data was analyzed using a one-way ANOVA with Tukey's multiple comparisons test. Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA and ACTIN protein levels from normoxic lysates of B16-F10 cells cultured alone or in the presence of the PHD2 inhibitor IOX2 (100µM) for 24h. Glut1, Pdk1, Mct4 and Vegfa transcript levels from B16-F10 cells cultured alone or in presence of the PHD2 inhibitor IOX2 (100µM) for 24h (n=4). Data were analyzed using a one-sample T-test against shCntl, except for Pdk1 shBNIP3 IOX2- which was analyzed using the non-parametric Wilcoxon test. Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA and ACTIN protein levels from normoxic lysates of B16-F10 cells cultured alone or in the presence of the iron chelator DFO (200µM) for 24h. Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, PHD2 and ACTIN protein levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against PHD2.
Figure 7A549-Luc cells transfected either with miR-515-5p precursor (miR), siRNAs to MARK4 (siMARK4) or the corresponding control sequences (C and NT, respectively) were injected in Scid mice through the tail vein. 4 weeks later, the mice were sacrificed, their lungs extracted and fixed prior to staining for Luciferase expression. (A) Representative pictures showing the presence of A549-Luc cells in the lungs of NT mice either as single cells (upper panel) or as a tumour mass (lower panel). (B) Percentage of the area covered by tumour cells on randomly observed fields of views. Quantification was performed using Photoshop CS4. Each spot corresponds to quantification of an individual 1 mm x 1.5 mm field. Transversal bar; Median. Statistics: Student t-test. *, p≤0.05. (C) Schematic summary. Increase in MARK4 expression is the main reason for miR-515-5p-down-regulated cells to show increased metastatic abilities in breast and lung cancer. The down-regulation of miR-515-5p de-represses MARK4 translation in individual cells from the primary tumour promoting their metastatic dissemination through the blood or lymphatic circulation and reducing patients' survival.
Grin2aS/S mice are hyperactive and show increased attention and lack of inhibition.Fig. 7a The total locomotor activity during the entire 2 h session in a novel environment was enhanced in Grin2aS/S mice. b Similarly, the exploratory behavior of Grin2aS/S mice remained high in all three successive 6 min exposure sessions to five novel objects (ITI = 4 min), as exemplified by traces of the animal's movement on the right. c (left) The time of first fall (Cliff Avoidance Reflex, CAR) was significantly reduced for Grin2aS/S and Grin2a+/S mice. c (right) In a 60-min session Grin2aS/S mice showed a significantly increased number of falls compared to wild-type mice. d (left) In the three-chamber sociability test Grin2aS/S mutants had a significantly reduced preference to explore the stimulus mouse over the object compared to Grin2a+/S and Grin2a+/+ littermates. d (right) Occupancy heat maps in the three-chamber sociability test show a representative example of a Grin2aS/S mouse with a reduced preference for another mouse versus an inanimate object. The social preference is visible for Grin2a+/+ and Grin2a+/S mice. The occupancy is color-coded separately for each group and translates to a % given in white numbers on the key for each genotype. f During the sample run in the Y-maze Grin2aS/S mice made significantly more total arm entries. In the test run, Grin2a+/+ and Grins2a+/S showed a preference for the unexplored novel Y-maze arm, whereas Grin2aS/S mice showed a lack of novelty preference. The numbers of mice are shown in brackets or below the bars of bar graphs. Chance levels (Ch.) are indicated by dashed lines. Error bars represent mean ± SEM (for statistics: Supplementary Statistics to Fig. 7).
TIP30 interacts with eEF1A.Figure 3(A) Western blot analysis of GST-pulldown assays with GST-TIP30 fragments of the indicated length (as: amino acids from N-terminus) with Myc-tagged binding partners. (B) Western blot analysis of co-immunoprecipitation (IP) from HEK cells transfected with GST-TIP30 and eEF1A1-Myc. (C) Confocal microscopy images of neonatal rat cardiomyocytes (NRCM) stained with antibodies for endogenous TIP30 (green) and endogenous eEF1A1 (red). DAPI: blue. (Scale bar: 20µm). (D) Microscopy images of isolated neonatal rat cardiomyocytes and subsequent Proximity Ligation Assay (PLA). Red: endogenous TIP30/eEF1A1 interaction; Blue: DAPI. Control cells were stained for TIP30/Myc-Tag interaction (scale bar: 50µm). (E) Western blot analysis of co-immunoprecipitation (IP) for endogenous TIP30 and eEF1A1 protein in NRCM after stimulation with phenylephrine for 24 h. IgG HC - High chain of IgG molecule. (F) Scheme showing the structure of TIP30-His deletion mutants that were used in GST-pulldown assays in (G). The C-terminal His-tag is highlighted in blue. Western blot analysis of GST-pulldown assays with GST or GST-tagged eEF1A1 (GST-eEF1A1) and TIP30-His full length protein (TIP30-His) and TIP30 deletion mutants ΔN25 (TIP30-ΔN25-His), ΔN52 (TIP30-ΔN52-His), ΔC15 (TIP30-ΔC15-His) and Δ102-107 (TIP30-ΔN102-104-His, G) Western blot analysis of GST-tagged eEF1A1 and GST-Control (H). (I) Scheme depicting eEF1A domains, their different binding patterns for GDP/GTP or eEF1B2 and the His-tagged eEF1A1 mutants. Western blot analysis of GST-pulldown assays with GST-TIP30 and the indicated His-tagged eEF1A1 mutants are shown. (J) Quantitative Real-Time PCR analysis of eEF1A1 and eEF1A2 mRNA abundance in hearts from neonatal mice, adult wild-type mice (Adult) and adult wild-type mice 2 weeks after TAC surgery (Adult TAC, N=3 to 4 mice/group). *P<0.05, ***P<0.001. One-way ANOVA with Sidak´s multiple comparisons test. (K) Western blot analysis for eEF1A1, eEF1A2 and GAPDH in hearts from wild-type mice at the age of 7 days (7d) and 8 weeks (8w) as well as 2 weeks (TAC 2w) after TAC surgery. (L) Western blot analysis of co-immunoprecipitation (IP) with GST-TIP30 and eEF1A2-His.
Figure 1(A) Kaplan-Meier survival curves (n = 12 - 16). (B) Heart gross morphology. (C) Body weight; (D) Heart weight and (E) Heart-to-body weight ratio (n = 28-40). (F) Relative expression levels of cardiac hypertrophy markers (Nppa and Nppb) (n = 5). Bars represent mean ± S.E.M. (Student's t test; **p < 0.01, ****p < 0.0001). (G) Enzyme histochemistry double staining for COX and SDH activities. (n = 4). (H) Assessment of cardiac fibrosis by Masson's trichrome staining (n = 4). White scale bars, 100μm; black scale bars, 1 mm.
Unless otherwise stated, all experiments were repeated three times and data shown are representative.figf3(a) WT MEFs were cultured with or without amino acids. ATG14L-associated Beclin-1 was immunoprecipitated and treated with lambda phosphatase treatment (PPase) as indicated. Western blotting (immunoblotting, IB) was performed with the indicated antibodies. Beclin-1 Ser 14 phosphorylation was quantified (shown under top panel) and normalized to total Beclin-1. (b) WT MEFs were starved for the indicated times. Beclin-1 was immunopurified by ATG14L immunoprecipitation and immunoblotted as indicated (top panels). Whole-cell lysates were immunoblotted with pULK1 S757 (mTORC1-target site), pS6K and ULK1 antibodies (bottom panels). Two unique experiments were performed. (c) WT or FIP200−/− MEFs were incubated under nutrient-rich, amino-acid-deprived or Torin-1 (+T, an mTOR inhibitor) conditions. Beclin-1 was purified and immunoblotted as in Fig. 3b. (d) WT or ULK-deficient MEFs were incubated with or without amino acids. Beclin-1 was purified and immunoblotted as in a. Two unique experiments were performed. Uncropped images of blots are shown in Supplementary Fig. S4.
Figure 1A. Endogenous Cyclin B1-YFP localisation during mitosis live in RPE1 cells (still from Movie EV1). Immunofluorescence images (B) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC. C. Immunofluorescence quantifications (C) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC. Each dot represents a kinetochore, data is from 40 kinetochore pairs (13 cells, max 5 kinetochore pairs/cell). D,E. Quantification of relative kinetochore intensities of Cyclin B1 and MAD1 in nocodazole-arrested cells (noco) treated with the MPS1 inhibitors, AZ-3146 (5μM) or Reversine (500nM), either before (D) or after (E) mitotic entry. F. Immunofluorescence images of LacI-MAD1 and Cyclin B1 in U2OS cells containing a LacO arrays on chromosome 1 G. Live imaging of Cyclin B1-mCherry (CycB1-mCh) in LacO-U2OS cells transfected with LacI-MAD1-FL (full length: aa1-718) or various LacI-MAD1 truncations (amino acid numbers indicated). H,I. Immunofluorescence images (H) and quantifications (I) of Cyclin B1 and MAD1 kinetochore levels in control (MAD1-WT) or MAD1β HeLa cells (two independent clones: C13 or C24) treated with nocodazole. J,K. Immunofluorescence images (J) and quantification (K) of Cyclin B1 and MAD1 kinetochore localisation in doxycycline-inducible MAD1α and β knockouts treated with or without dox for 10 days and then arrested in nocodazole. Cells were selected that had full MAD1 knockout in the doxycycline treatment (this constituted approximately 30% of cells). L. Relative kinetochore volumes occupied by Cyclin B1 and MAD1 (relative to CenpC) in nocodazole-arrested MAD1α and MAD1β cells (calculated from experiments shown in (H,I).
Defect in nuclear import delays INQ formation A, B S.Figure 4A, B S. cerevisiae hsp42Δ and hsp42Δ nup42Δ cells expressing mCherry‐VHL (red) were grown at 30°C and shifted to 37°C in the presence of MG132. Changes in protein localization were monitored. Scale bars, 2 μm. The total number of mCherry‐VHL foci per cell was determined. C Dynamic exchange of a misfolded model protein between cytosol and nucleus. FLIP measurements of mCherry‐VHL were performed in S. cerevisiae wild‐type cells at 30°C. Nuclear or cytosolic areas were bleached, and loss of fluorescence intensities in non‐bleached compartments was determined. Bleaching and acquisition controls are given. Error bars: SEM.
Figure 5A. Schematic for replication fork protection assays in (B) and (C). B. Replication fork degradation (IdU/CldU ratio) in U2OS cells transfected with indicated siRNAs and processed as in (A) (bars, mean; n=200 fibers, pooled from two independent experiments, per condition). C. As in (B), except fork degradation was analyzed in U2OS/GFP-RADX cell lines. D. U2OS or U2OS/GFP-RADX cells transfected with indicated siRNAs and exposed to HU for 4 h were co-immunostained with RPA1 and γ-H2AX antibodies and analyzed by QIBC. Proportion of cells displaying maximal RPA chromatin loading accompanied by H2AX hyperphosphorylation (Fig EV5A), reflecting replication fork catastrophe [17], is indicated (bars, mean; n=5 independent experiments (≥3000 cells analyzed per condition); *P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001, unpaired t-test). E. As in (D), but using U2OS or U2OS/Super-RPA cells exposed or not to HU (n=6 independent experiments). See also Fig EV5B. F. Clonogenic survival of U2OS cells transfected with indicated siRNAs and subjected to different CPT doses for 24 h (mean±SEM;n=3 independent experiments). LQ (Linear Quadratic) model was fitted to the fractional survival data, using non-linear least square method. Overlap between confidence intervals of the fitting coefficients was used to evaluate the statistical difference between cell survival after siRADX and siCTRL treatment (*No overlap of 95% confidence interval with siCTRL). G. As in (F), except cells were treated with indicated doses of HU (mean±SEM;n=3 independent experiments). H. As in (F), using U2OS and U2OS/Super-RPA cells (mean±SEM;n=3 independent experiments).
Figure 1A. Schematic workflow of the co-culture experiment in which AECs were subjected to apical TGFβ1 stimulation for 72 h in the co-culture system. Mono-cultures were employed as negative controls. B. Representative E-cadherin (E-cad) and DAPI immunofluorescence images of AECs treated apically with TGFβ1 or vehicle control after 72 h in the co-culture system (scale bars 25 µm). C. ELISA analysis shows decreased E-cad levels in TGFβ1-treated AECs after 72 h in the mono- and co-culture systems (n = 6 biological replicates, mean + s.d., ***p < 0.001, ANOVA/Tukey´s). D. Representative αSMA and DAPI staining of healthy and IPF-derived MCs from the mono- and co-culture systems after 72 h apical treatment with TGFβ1. Note an increase in αSMA levels only in the presence of AECs and further enhanced in IPF-MCs (scale bars 100 µm). E. ELISA analysis shows increased αSMA levels in a TGFβ1 dose-dependent manner from MCs and IPF-MCs in the co-culture system. Note a stronger αSMA response in IPF-MCs (mean + s.d., n = 3 biological replicates from 6 MCs and 6 IPF-MCs donors, **p = 0.0014, ****p < 0.0001, Nonlinear Regression followed by ANOVA/Sidak's). F. Schematic workflow of the substitution system in which AECs were subjected to TGFβ1 stimulation for 3 h in co-culture with MCs, after which (1) MCs or (2) AECs were replaced with untreated counterparts or (3) both fresh media and untreated MCs for a further 72 h. G, H. 72 h after substitutions in the co-culture, αSMA and E-cad levels of MCs and AECs, respectively, were measured with ELISA and normalised to the control. Note replacement of injured AECs with untreated AECs or replacement of media and MCs attenuates αSMA expression and increases E-cad levels (mean + s.d., ns = non-significant, ***p < 0.001, **p < 0.01, n = 3 biological replicates with 6 MCs donors, repeated measurement ANOVA for MCs or ordinary ANOVA for AECs with Dunnett´s posthoc test).
Figure 6(A-C) Ribbon views of the S. cerevisiae Hrr251-394:Mam187-191 structure (form 1), CK1-7 bound S. cerevisiae Hrr251-394, and ADP-bound C. glabrata Hrr251-403 (formate condition), with Hrr25 colored according to main-chain B-factor from low (purple) to cyan (high). For each, coloring is normalized to correspond to the average of the 20 lowest (purple) or highest (cyan) main-chain B-factors in Hrr25. Mam1 (panel A) is colored in light gray. (D) Overall main-chain B-factors for the N-lobe (residues 1-85) of four structures of Hrr25 (both C. glabrata and S. cerevisiae) and the three crystallographically-unique views of the S. cerevisiae Hrr25:Mam1 complex. Values are normalized to the average main-chain B-factor for the entire Hrr25 chain (dotted line at 1.0). P-value=0.004 (student's T-test). See Figure EV4 for residue-by-residue B-factor plots for each structure.
Efficient chemical inhibition of TRF1 telomere binding by compounds ETP-47228 and ETP-47037 in mouse lung adenocarcinoma-derived cells.Figure 7Quantification of TRF1 levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with 10 μM ETP-47228 (24 h), and with 10 μM ETP-47037 (48 h). Representative images are shown to the right. Quantification of γH2AX levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with ETP-47228 (24 h), and with ETP-47037 (48 h). Representative images are shown to the right. Quantification of telomere-induced foci (TIFs) by double immunofluorescence with anti-RAP1 and anti-γH2AX antibodies. Representative images are shown to the right. White arrowheads: colocalization of γH2AX and RAP1. Effect of different ETP-47228 and ETP-47037 concentrations during 24 h on proliferation in lung tumor-derived cell line relative to the growth of DMSO-treated cells. Tumor growth quantification in allograft model ETP-47037 or with ETP-47228.
Figure 2Schematic representation of the experimental induction of CARD14E138A transgene using tamoxifen. WT = K14creERTg/+ Rosa26+/+, ieCARD14E138A = K14creERTg/+ Rosa26LSL-CARD14-E138A/+ Changes in relative body weight and ear thickness upon CARD14E138A induction with tamoxifen. Malt1EKO = Malt1fl/fl. The combined results of five independent experiments are shown (Malt1+/+ WT n=17, Malt1fl/fl WT n=8, Malt1+/+ ieCARD14E138A n=18, Malt1fl/fl ieCARD14E138A n=12). Representative H&E-stained histological sections of ear tissue of tamoxifen-treated mice (Scale bar represents 200 μm). Arrowheads indicate infiltrates of immune cells. Epidermal thickness measured on ear sections. Each symbol represents the mean of at least ten measurements for each ear; the line represents the mean value. The combined results of three independent experiments are shown. Analysis of infiltrating immune cells in the ears of tamoxifen-treated mice using flow cytometry. Cell count of neutrophils (CD45+ CD3/CD19- CD64- CD11b+ Ly6G+), eosinophils (CD45+ CD3/CD19- CD64- Ly6G- SiglecF+), T cells (CD45+ CD3/CD19+, MHCII-), DCs (CD45+ CD3/CD19- CD64- MHCII+CD11c+) in single cell suspensions of the ear (n≥4). mRNA expression levels of the indicated genes in ears relative to reference genes (Hprt1, Rpl13a and Tbp1). Each symbol represents one mouse; the line represents the mean value (n≥3).
Chromatin accessibility and epigenetic signature of control and GS∆44 myoblasts.Figure 3A DNA accessibility (ATAC-sequencing) and chromatin marks (ChIP-seq) signals in the Celf2 region (+/- 50kb) using the UCSC genome browser. ATAC-seq peaks called in the two conditions are reported below each track. The differential peaks specific for one of the two conditions are reported in the tracks ATACseq_specific peaks_WT and ATACseq_specific peaks_GS∆44. ChIP-seq signals for H3K4me3, H3K27ac and H3K27me3 in WT and GSΔ44 myoblasts (WT myoblasts in light orange; GSΔ44 myoblasts in light blue). ChIP-seq for MyoD in control cells was obtained from (MacQuarrie et al, 2013) (GSM1218849). Orange boxes highlight the regions containing the first exon of each isoform (a, b and c). B Pie charts showing ATAC-seq peaks' genomic distribution (blue: promoter region, grey: gene bodies, white: intergenic regions). C Pie charts showing genomic distribution of ChIP-seq peaks (H3K4me3, H3K27ac and H3K27me3) (blue: promoter region, grey: gene bodies, white: intergenic regions). D Chromatins extracted from Control (WT) and GS∆44 myoblasts were immunoprecipitated with anti-MyoD and control IgG antibodies. Regions corresponding to the MyoD binding sites present in Peak A (see Figure EV3D) and in the MAFA and LSTZ2 genes were analysed by qPCR. Fold enrichments of PeakA over MAFA or LSTZ2 immunoprecipitation (IP) are plotted for WT and GS∆44 samples. The mean ± SEM of triplicates from one representative among three experiment is shown. Statistical significance of differences between means was assessed by a two-tailed t-test and P<0.05 was considered significant. *P=0.0339, **P=0.0067.
Figure 2Chromatin marks associated with promoters are observed in hypomethylated regions without young LINE1 retrotransposons (HMR_-L1s). Metaplots show the average ChIP-seq signals across HMR_-L1s, transcription start sites (TSSs), and randomly selected sites. The signals were normalized by either H3 or input signal. The results shown in heat maps were not normalized by H3 or input. The initiation form of Pol2 with an unphosphorylated carboxy terminal domain (Pol2 null-P CTD) is highly enriched in HMR_-L1s.
3D reconstruction of Dln1 octamer.Figure 5A. Negative-stain electron microscopy image of Dln1M5 in presence of liposomes.B. Side views of Dln1M5 oligomers attached to the membrane of liposomes. The inset represents the respective class averages. C&amp;amp;D. Transmission electron microscope micrograph of dispersed ring-like structures of Dln1M5 or Dln1D135A oligomers on lipid monolayer. The inset represents a few 2D class averages showing the octamer pattern from some individual rings. C&amp;amp;D. Transmission electron microscope micrograph of dispersed ring-like structures of Dln1M5 or Dln1D135A oligomers on lipid monolayer. The inset represents a few 2D class averages showing the octamer pattern from some individual rings. E. EM picture of a negatively stained 2D crystal of Dln1M5 on the lipid monolayer. The inset represents the computed diffraction pattern Fourier transform of the image.F. Projection map with a P4 symmetry. G. The top, side and cut-open views of the 3D reconstruction of Dln1M5 octamer at 20 Å resolution are shown as gray surface representation. The surface of membrane is indicated by dashed lines. The scale bar is 10 nm.
Figure 9Figure 9. Functional domains of Syt11 in endocytosisA. Diagram of the mutant forms of Syt11 used for rescue experiments.B-D. Statistics of FM4-64 (5 min), Tf, and large dextran (40-KD) uptake into KD neurons with or without rescue by indicated forms of Syt11.All data are presented as mean ± s.e.m. of 3 independent experiments. One-way ANOVA, values labeled with different letters (a, b, and c) are significantly different from each other, ***P < 0.001. Student's t-test for the comparison between KD and control cells in (B-D), **P < 0.01, ***P < 0.001.
Figure 3A) Schematic of proteomics experimental pipeline. Lung fibroblasts from 8 mice (4 WT, 4 CKO) were isolated and expanded. Samples were taken 12 hours apart, starting 24h after a medium change ("Experimental time 0 h"). B) Bar chart reports the number and % of detected proteins showing a significant difference in abundance for the listed comparisons by two-way ANOVA correcting for multiple comparisons, controlling for false discovery rate by the Benjamini, Krieger and Yekutieli method (q=0.05). C) Most of the proteins whose abundance changed significantly over time differed between WT and CKO cells. D-E) Proteins with significant abundance change over time had no significant difference in mean absolute fold change between WT and CKO cells (D, p=0.47), but significantly greater variance in absolute fold change between independent CKO biological replicates than between their WT counterparts (E, **p=0.0013); Kolmogorov-Smirmov test (n=4). Continuous lines = median, dotted lines = quartiles. F) Proteins with significant differences in overall abundance between CKO and WT cells were more likely to show genotype-dependent differences in temporal variation (Fisher's exact test, p<0.0001), number of proteins in each group reported. G) Absolute fold changes were calculated from maximum/minimum for all protein abundances and probability densities plotted. There were significant differences between WT vs time fold changes and WT vs CKO fold changes (****p<0.0001). There was no significant difference between WT vs time fold changes and CKO vs time fold changes (p=0.930), Kruskal-Wallis test with Dunnett's multiple comparisons test, p-values adjusted with Benjamini-Hochberg method. H) Data was extracted from Mauvoisin et al. (2014), where the proteome was quantified in WT and CKO mouse livers extracted under diurnal conditions. Absolute fold change was calculated from maximum/minimum protein abundances. Probability density distributions for fold-change are plotted. Kolmogorov-Smirnov test: ****p<0.0001.