EMBO/sd-character-level-ner huggingface dataset:
Figure 5A. Schematic of SLAMseq workflow. Cells were pretreated for 30 min with LSD1 inhibitor (100 nM GSK-LSD1) and newly synthesized RNA was subsequently labelled for 1 or 6h with 4-thiouridine (4sU). RNA was extracted and alkylated and subjected to 3'-sequencing. B. MA-plot of the mRNA changes detected by SLAMseq after 1h and 6h of 4sU labelling. Significantly up- and downregulated genes (FDR≤0.05; abs(log2FC)≥0.5) are marked in red and blue, respectively. Genes belonging to neuronal differentiation (GO:0048666) are labelled. FC, fold-change; CMP, counts per million. C. Pathway-enrichment analysis for upregulated (FDR≤0.05; log2FC≥0.5) direct transcriptional targets of LSD1 in Fig 5B. D. Circos plots depicting the transcriptional changes of genes up- (up) and down-regulated (down) upon LSD1i after 1h, 6h, 24h and 6d. DE, differentially expressed. E. Metascape analysis of upregulated genes for enriched pathway terms in genes identified after 6h of 4SU-labeling. Major pathway clusters are labelled. F. Schematic of Merkel cell extraction and Merkel cell signature generation. DE, differential expression; FDR, false discovery rate; log2FC, log2 fold-change. G. Pathway enrichment analysis (GO:BP) of genes upregulated in Merkel cell signature. H. Gene set enrichment analysis (GSEA) of the generated Merkel cell signature on the data in Fig 4B. NES, normalized enrichment score; FDR, false discovery rate.
Figure 6A. EMSA showing IDAP2 binding to DNA probes and competition by unlabeled DNA probes. B, C. The effects of the W19E and W48E mutations on IDAP2 binding to DNA probes. Asterisks indicate non-specific bands. B, C. The effects of the W19E and W48E mutations on IDAP2 binding to DNA probes. Asterisks indicate non-specific bands. D. Complementation assay showing that wild-type, but not the mutant form (W48E), of IDAP2-3 Flag can restore LUC expression in idap2-1. E. Analysis of the DNA methylation level at the DT-75 locus by chop-PCR. McrBC is a methylation-sensitive restriction enzyme, and DNA hypermethylation results in a reduced level of the PCR product. Undigested DNA was amplified as a control.
Figure 5A. T6SS secretion assay of A. tumefaciens wild type C58, ΔtssL, and various single, double, and triple toxin-immunity deletion strains. B. T6SS secretion assay of A. tumefaciens wild type C58, ΔtssL, Δtdei, and Δ3TIs harboring the indicated plasmids.
Figure 2CSF PGRN as a function of EYO in mutation carriers (MC, red) and non-carriers (NC, blue). The solid lines indicate the regression line for each of the groups and the 95% confidence interval (CI) calculated by a linear model adjusting by gender. The interaction term of mutation status and EYO is significant (P = 0.041), also when including PGRN outliers and participants with EYO > +20 (P = 0.030). Individual data points are not displayed to prevent disclosure of mutation status. The graph depicts the standardised differences of CSF PGRN between MCs and NCs as a function of EYO, in the context of other biomarker and cognitive changes. The curves were generated by the linear model that best fit each marker (see statistics section and Appendix Table S2). CSF PGRN is significantly increased in MC compared to NC 10 years before the expected symptom onset (shadowed area) after brain amyloidosis and brain injury (as measured by CSF T-tau) have started, and shortly before CSF sTREM2 starts to increase. Abbreviations: Aβ1-42: amyloid-β 42; CSF, cerebrospinal fluid; MC, mutation carriers; MMSE, Mini-Mental State Examination; NC, non-carriers; T-tau, total tau.
dEsyt is dispensable for phototransduction.Figure 2(A) Representative ERG trace from dark reared 0-1 day old flies of the indicated genotype stimulated by 2s flash of green light. The duration of the stimulating light is shown. Y-axis shows the amplitude of the ERG response. X-axis shows the duration of the recording. (B) Graph showing ERG amplitude normalized to body weight. Y-axis shows the ratio of ERG amplitude of individual flies to their body weight. X-axis indicates genotypes. Each data point represents an individual fly tested. Error bar represents s.e.m. (C) Quantification of the mean fluorescence intensity of the deep pseudopupil formed by the PIP2 probe PH-GFP in the one day old flies of the indicated genotypes (n=10). (D) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors. The fluorescence of the PH-GFP probe is depicted. The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light (a-f) used for image capture depicted. Representative deep pseudopupil images acquired at specified time points are depicted. Genotypes as indicated. (E) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time, X-axis represents the genotypes, Y-axis represent intensity normalised to the intensity of the first image. (n=10 biological replicates). Error bars represent s.e.m. (F) Quantification of the time course of retinal degeneration. 10 ommatidia from 5 separate flies of each genotype were scored and plotted. Y-axis is the number of intact rhabdomeres/ommatidium. The maximum value possible is 7. X-axis is the age of the flies post eclosion. (G) Representative optical neutralization images showing rhabdomere integrity of the indicated genotypes. Rearing conditions and the age of the flies are indicated on top. (*CL- Constant Light) (H) Representative ON images showing the rescue of rhabdomere structural integrity in dEsytKO mutants on expressing the dEsyt::mCherry transgene. Genotypes of the flies are indicated on the left. Rearing conditions and the age of the flies are indicated on top.
The localization of both HXT2 mRNA and Hxt2 protein is cell-cycle dependent.Figure 1A The distribution of the GFP-tagged Hxt2 changes during the cell division. In small and medium budded cells (white arrowheads), Hxt2 is mainly present in the membrane of the mother cell. In large budded cells, Hxt2 is distributed over the entire plasma membrane (yellow arrowheads). B HXT2 mRNA localization correlate with that of the protein. FISH with an HXT2-specific probe. Early in the cell cycle the mRNA is predominantly found in the mother cell (white arrowheads) while it is equally distributed after meta-to-anaphase transition (MAT) (yellow arrowheads). C Quantification of (B). The fluorescence intensities from mother and bud were independently measured and the ratio of the signal from the mother over the signal from the bud was calculated. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Three independent experiments with 50 mother cells and 50 buds per experiment were analyzed. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively. D Changes of HXT2 mRNA localization appear to be independent of the cytoskeleton. Cells were either treated for 15 min with 30 mg/ml latrunculin A or with 30 mg/ml benomyl. White arrowheads point to small-budded and yellow arrowheads to large budded cells. E Quantification of (D) of three in dependent experiments with at least 50 cells each. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively.
Figure 3A. Representative images and drawings of Golgi-stainedhippocampalCA1pyramidal neurons from 4 weeks-old wild type and Lrig1-null littermate mice. Scale bar, 15 m.B. Quantification of the number of primary dendrites and branching of apical and basal dendritic arbors of hippocampalCA1pyramidal neurons from 4-5 weeks-old control (wild type/heterozygous) and Lrig1-null littermate mice. The results are shown as mean SEM of independent determinations performed in n=4 mice of each genotype. *p<0.05, **p<0.001 by Students t test. NS, not significant.C. Cummulative dendrite crossings of concentric circles of increasing radius (10 μm ring interval) centering the reference point at the cell body. These values were obtained by Sholl analysis and represent the summatory of the dendritic crossings registered within the first 60 m closest to the soma. The results are shown as mean SEM of independent determinations performed in n=4 mice of each genotype. *p<0.05 by Students t test.D. Sholl analysis of apical and basal dendritic arbors of hippocampalCA1pyramidal neurons from 4-5 weeks-old control (wild type/heterozygous) and Lrig1-null littermate mice. The results are shown as mean SEM. *p<0.05, **p<0.001 by two-way ANOVA followed by Bonferroni multiple comparisons test.Quantifications showed in B-D were performed in n=60 neurons from 4 wild type/heterozygous mice and 4 Lrig1-null littermate mice (n=4).
Figure 3HEK 293T cells were transfected with doxycycline-inducible DmrB-caspase-8 and the indicated GSDMD constructs. Cells were stimulated with doxycycline (10 μg/ml) for 18 h to induce DmrB-caspase-8 expression and exposed to B/B homodimerizer (12.5 nM) for another 2 h to activate caspase-8. Mixed supernatant and extracts were analysed by immunoblot. Immortalized Gsdmd-/- BDMDs expressing GSDMDWT and GSDMDD88A were costimulated with TNF (100 ng/ml) and SM for 6 h and LDH release was quantified. Immortalized Gsdmd-/- BDMDs expressing GSDMDWT and GSDMDD88A were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with LCL161 (1 μM) for 24 h and LDH release was quantified. BMDMs were costimulated with TNF (100 ng/ml) and TAK1i for 4 h and (D) mixed supernatant and extracts were analysed by immunoblot BMDMs were costimulated with TNF (100 ng/ml) and TAK1i for 4 h and LDH release was quantified at the indicated time points.
Model of the Cvt pathway.Figure 6 Figure 6: Model of the Cvt pathway. (A) prApe1 the major Cvt cargo forms homo-oligomers as dodecamers that (B) assemble into higher-order assemblies mediated by the N-terminal propeptide. (C) These assemblies are capable of binding Atg19 that forms a trimer in solution. (D) Ams1, which forms a homo-oligomer as a tetramer, joins the Cvt assembly by binding to Atg19 via a site that is distinct from the Ape1 binding site. (E) prApe1 dodecamers, secondary cargo enzymes such as Ams1 tetramers and Atg19 trimers are the main constituents of the Cvt aggregate in the cytosol. (F) The Cvt aggregate becomes enwrapped by a double membrane via Atg8 and Atg19 interaction to form (G) the Cvt vesicle. Cvt assembly stages in (E), (F) and (G) are drawn respecting the determined average size of the vesicles, the dimensions and stoichiometry of the major cargo prApe1 and secondary cargo Ams1 found in this article. (H) After fusion with the vacuole, prApe1 is subjected to maturation while maintaining the dodecamer organization. (I) Ams1 does not undergo a maturation process in the vacuole.
Figure 4A Dose-dependent viability assays of PDTCs derived from breast cancer samples (Bruna et al., 2016) treated with pyridostatin (PDS) at the indicated doses. Values from n = 2 independent experiments are shown. AB521: ER-negative tumour, no BRCA1 alteration; VHIO179: tumour with BRCA1 germline mutation and MAD2L2 inactivating mutation (olaparib-resistant). B VHIO179 PDTXs were grafted into CB17-SCID female mice. Pyridostatin was administered intravenously (7.5 mg/kg/day) over the indicated periods of time. Vertical dotted line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 7 mice. Error bars represent SEM. P values were calculated between treated and untreated tumours at day 24, using an unpaired two-tailed t-test. ****, P ≤ 0.0001.
Cell size, cell growth and apoptosis in TIMM50 mutant fibroblasts A Cell size in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose.Figure 6A Cell size in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Data are shown as mean ± SD, n = 5 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001. B Growth curves of control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Cell growth was monitored continuously by the Incucyte live cell imager (Essen Bioscience). One of the three independent experiments carried out is presented. Data are shown as mean of three technical replicates ± SD. C Plot of Annexin V intensity against propidium iodine signal in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Cells in the late stage of apoptosis are boxed in the top right corner. D Quantification of the number of late apoptotic cells in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Data are shown as mean ± SD, n = 5 or 4 biological replicates in the case of transduced cells; ∗*∗p < 0.001.
Figure 5(A) C-09.10 short-term culture cells were infected with retroviruses expressing ZEB1. Western blot analyses of ZEB1 and p75. GAPDH was used as a loading control. (B) Quantitative PCR analyses of MITF and p75 upon ZEB1 expression. mRNA expression levels are represented relatively to control cells (mean ± SD, n=3, Student's t-test). (C) Soft agar colony formation assay following ZEB1 expression. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test). (D) FACS analyses of p75 cell surface expression upon ZEB1 overexpression. Bar chart representing the mean percentage of p75-high, int, low and negative cells from 2 independent experiments (Fisher's exact test). (E) Clonogenic assay +/- PLX4032 (100 nM) treatment for 10 days. The graph represents the mean number of colonies in 3 independent experiments (Student's t-test).
Domain analysis of Atg14L structure.Figure 2.(a) HEK293 cells were simultaneously transfected with four plasmids harboring One-Strep-Flag (OSF)-tagged Atg14L, Beclin-1, hVps34, and hVps15, and Atg14L was pulled down using Strep-Tactin Sepharose beads. Empty plasmid was used as control instead of Atg14L. The precipitates were subjected to SDS-PAGE and Coomassie brilliant blue staining. (b) Diagram of Atg14L deletion mutant construction. (c) HEK293 cells transiently expressing each construct were subjected to coimmunoprecipitation analysis with the anti-GFP antibody. Each precipitant was subjected to immunoblotting with the indicated antibodies. IP, immunoprecipitation. (d) Alignment of N-terminal conserved region of Atg14 orthologues. Conserved cysteine repeats are boxed. The degree of sequence conservation is denoted: asterisks indicate a perfect match among interspecies, periods indicate mostly conserved, and colons indicate more conserved than the periods. Homo sapiens (Hs), Mus musculus (Mm), Gallus gallus (Gg), Danio rerio (Dr), Drosophila melanogaster (Dm), Candida glabrata (Cg), and Saccharomyces cerevisiae (Sc) are shown. (e) NRK cells were transiently transfected with adenovirus expressing GFP-Atg14L or GFP-Atg14L4C4A and fixed under nutrient-rich conditions. The cells were stained with anticalnexin antibody as an ER membrane marker. Higher magnification of the boxed areas is shown below. Bars, 10 µm.
Ref(2)P localizes in protein aggregates in old flies during normal aging.Figure 2.(A-D) Electron micrographs of adult brain of old wild-type flies. Ref(2) P localizes in electron-dense structures ranging from 50 nm to 1 μm in diameter that are surrounded (arrows) or not (arrowhead) by a limiting membrane. (E and F) Electron micrographs of wild-type brain of old flies demonstrating that Ref(2) P forms shell-like structures that surround aggregated filamentous material (arrows). Bars: (A-C) 50 nm; (D) 100 nm; (E and F) 200 nm.
Figure 1A. Schematic depiction of bronchioalveolar cell types and strategy for selective in vivo manipulation of dual-marker expressing target cells. Data information: Npu: Nostoc punctiforme, Ssp: Synechocystis sp., CDS: coding sequence. BASCs: bronchioalveolar stem cells, BADJ: bronchioalveolar duct junction, NLS: nuclear localization signal. B. Split-effector construct design. Data information: Npu: Nostoc punctiforme, Ssp: Synechocystis sp., CDS: coding sequence. BASCs: bronchioalveolar stem cells, BADJ: bronchioalveolar duct junction, NLS: nuclear localization signal. C. Quantification of Cre activity by dual luciferase assays (mean±SD, n=3). Data information Npu: Nostoc punctiforme, Ssp: Synechocystis sp., CDS: coding sequence. D. Quantification of tTA activity by dual luciferase assays (mean±SD, n=3 (N and C) or n=7 (N/C)). Data information: Npu: Nostoc punctiforme CDS: coding sequence.
Figure 1Expression of ERα in control (shCtrl) and knockdown (shERα) BM67 cells was determined by western blotting (β-actin served as a loading control). Gene expression was determined using polysome-profiling which quantifies both total mRNA and efficiently translated polysome-associated mRNA (n = 3). Distributions (quantified by kernel density estimation) of p-values for differential expression (shERα vs. shCtrl BM67 cells) using data from polysome-associated mRNA (orange), cytosolic mRNA (purple) or from analysis of changes in translational efficiency (i.e. changes in polysome-associated mRNA not paralleled by changes in cytosolic mRNA) leading to altered protein levels (red; a higher density of low p-values indicates a higher frequency of changes in gene expression). Scatterplot of polysome-associated mRNA vs. cytosolic mRNA log2 fold-changes (shERα vs. shCtrl). Areas of the plot are colored according to the density of data points (genes; dark blue corresponds to areas with many genes Same plot as in (B) but including a blue density of p-values from the analysis of translational offsetting. A scatter plot similar to (C) but where genes are colored according to their mode of regulation derived from anota2seq analysis. A relaxed threshold (p<0.05) was used to identify a set of transcripts regulated via translation, which did not pass thresholds used for identification of changes in mRNA abundance or translational offsetting (FDR<0.1). Confidence ellipses (level 0.7) are overlaid for each mode of regulation. A bar graph indicates the number of mRNAs regulated via each mode.
Figure 5A. Deconvolved inverted fluorescence micrographs of Chm7-GFP in the indicated strains. Scale bar is 5 μm. The percentage of cells ± SD with Chm7-GFP foci is indicated below each panel. B. Deconvolved fluorescence images of SINC-containing nuclei in vps4Δ and vps4Δpom152Δ cells expressing Chm7-GFP and Nup170-mCherry (green, red and merged images are shown). Scale bar is 1 μm. C. Plot of the proportion of nup clusters with Chm7-GFP enrichment in the indicated genetic backgrounds. Error bars are SD of the mean from 3 independent replicates. D. Deconvolved fluorescence micrograph showing the lack of colocalization of Chm7-GFP and clustered Nup170-mCherry in a nup133Δ strain. Scale bar is 1 μm. E. Plot of the total fluorescence/integrated density (in arbitrary units (A.U.)) of Chm7-GFP within (and outside of) the SINC. Error bars represent SD deviation of the mean from > 50 SINC or non-SINC accumulations pooled from 3 independent replicates. p values from un-paired student's T-tests with Welch's correction. **** is p ≤ 0.0001. F. Correlation of the total fluorescence intensity/integrated density of SINC-enriched Chm7-GFP and Nup170-mCherry within individual SINCs. Linear regression calculated from 150 SINCs pooled from 3 independent replicates; r is the linear correlation (Pearson's) coefficient.
Figure 6Left panel) H2A.Z.1 promotes the expression of several cell cycle genes whilst repressing the G1/M checkpoint proteins p21 and p27 thus favouring cell cycle progression. Upon downregulation of H2A.Z.1, the expression of these genes is altered leading to G1/S cell cycle arrest and cellular senescence. Right panel) H2A.Z.2, through its post-translational modifications, regulates the recruitment of several centromeric proteins and contributes to the regulation of the spindle assembly checkpoint. H2A.Z.2 depletion leads to a defective SAC, premature sister chromatid separation and chromosome segregation errors.
Figure 3(A) T47D-MTVL cells were treated with R5020 as indicated and subjected to ChIP assays with a-BRG1 or control IgG. Precipitated DNA was analysed by PCR for the presence of sequences corresponding to the BCAS1, KRT23 and IGFBP5. The histograms show the mean ± SD of 3 experiments performed in duplicate. * P-value < 0.05. (B) T47D-MTVL or T47DY (PRA-/PRB-) cells were treated or not with hormone and subjected to ChIP assays with antibodies to BRG1. * P-value < 0.05. The levels of PR and BRG1 were analyzed by western blotting using specific antibodies (right panel). (C) T47D-MTVL cells treated or not with hormone were lysed and immunoprecipitated either with a-HP1y specific antibody or mouse IgG. The immunoprecipitates (IP) were analyzed by immunoblotting with PRS294phos, PR, BRG1, BAF170, BAF57, KDM1 and HP1y specific antibodies. D) Protein-fractionation of hormone-treated T47D cell lysates by FPLC with Superose 6 sizing column was analyzed by immunoblot with BRG1 and BAF170 specific antibodies (upper panel). The BAF complex containing BRG1 and BAF170 as well as a smaller complex of BRG1 are indicated (upper panel). Then, IP of BRG1 in the BRG1 eluting fractions from the Superose 6 was performed followed by western blot with BRG1, BAF170, BAF155, LSD1 and HDAC1 (lower panel). The scheme with the protocol is shown (left panel). E) T47D-MTVL cells transfected with control or BRG1 siRNAs were treated with 10 nM R5020 as indicated; cDNA was generated and used as template for real time PCR with gene-specific primers. The histograms show the mean ± SD of 3 experiments performed in duplicate. * P-value < 0.05. The extent of BRG1 knockdown was monitored by RT-PCR (pink column) and western blot (right panel). BC: BCAS1, KR: KRT23 and IG: IGFBP5 genes. F) Cells were transfected with siRNAs and treated with 10 nM R5020 as indicated, and the levels of repressed genes was measured by RT-PCR. The extent of knockdown was analyzed by western blotting using specific antibodies (lower panel). * P-value < 0.05. (G) Cells were transfected with control or BRG1 and BRM (B/B) siRNAs and treated with 10 nM R5020 as indicated, and the levels of repressed genes was measured by RT-PCR. * P-value < 0.05. BC: BCAS1, KR: KRT23 and IG: IGFBP5 genes.