EMBO/sd-character-level-ner huggingface dataset:
Figure 3mRNA expression of ATF3 by qRT-PCR in control, ATF3 knockdown (KD), and ATF3 overexpression (OE) AGS cell lines generated using a PiggybacTM transposon system. Significance by one-way ANOVA, Dunnett post-hoc. Datapoint = mean of technical triplicates in a single mouse. n=3 mice. Error bars denote standard deviation. mRNA expression of RAB7B by qRT-PCR in AGS cell lines from 3A. Statistics and plotting as for panel A. Top - Immunofluorescence for RAB7 (red) and DAPI (blue, nuclei) in control, ATF3 knockdown (KD), and ATF3 overexpression (OE) AGS cells. Arrowhead indicates aggregated RAB7 vesicular formation similar to those observed in tissue. Bottom - Immunofluorescence for ATF3 (purple) and Cathepsin D (CTSD, green, lysosomes) in AGS cells. Scale bar, 20µm. Left - Immunofluorescence for anti-FLAG (RAB7B) (red) and DAPI (blue, nuclei) in ATF3 knockdown; RAB7B overexpression (ATF3-KD RAB7B-OE) AGS cells show large vesicular structures. Right - Immunofluorescence for anti-FLAG (RAB7B) (red) and DAPI (blue, nuclei) in ATF3 overexpression; RAB7B GTPase mutant (ATF3-OE RAB7BT22N) AGS cells fail to make vesicles. Scale bar, 20µm.
a) Immunoblot for LC3 in MOBs from WT or Cx43-null mice (Cx43−/−) treated with vehicle or with 18α glycyrrhetinic acid (18α GA).figf3(a) Immunoblot for LC3 in MOBs from WT or Cx43-null mice (Cx43−/−) treated with vehicle or with 18α glycyrrhetinic acid (18α GA). Glyceraldehyde-3-dehydrogenae (GPD) is shown as the loading control. Levels of LC3-II (left) and LC3-II flux (right; n = 3 independent experiments). (b) Immunofluorescence for LC3 in MOBs treated with vehicle or 18α glycyrrhetinic acid. Where indicated, chloroquine (CQ) was added to block lysosomal degradation. Bottom: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment). (c) Immunofluorescence for LC3 in WT or Cx43−/− MOBs treated with the indicated kinase inhibitors for 2 h. Insets: Higher magnification images in inverted black and white. (d) Endogenous LC3 puncta in the same cells shown in c (n = 3 wells, 3 independent experiments, >50 cells per experiment). *, differences from WT; §, differences from untreated. (e) LC3 puncta in Cx43−/− MOBs transfected with constructs coding for different forms of Cx43 (full length, CT, ΔCT258 or ΔCT245) tagged as indicated in the scheme. Left: Representative images. Merged fields (left) and LC3 channel in reverse black and white (right) are shown (images of GFP-Cx43 are shown in Fig. 2f). Right: Quantification of the average number of LC3 puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment). (f,g) Immunoblot for the indicated proteins of the immunoprecipitates (IP) of Atg16 from MEFs transfected with GFP-tagged full-length (FL-) Cx43 or Flag-tagged C terminus (CT-) Cx43 (f) or ΔCT258-Cx43 (g) I: input; FT: flow through. GAPDH and GFP alone were used as negative controls for immunoprecipitation and Atg5 as the positive control. All values are mean + s.e.m. Differences from the control were significant for (*, §)P 0.01 and (**, §§)P 0.001. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.
The N‐terminus of Noc contains an amphipathic helix A AMPHIPASEEK prediction result for Bacillus subtilis Noc.Figure 2A AMPHIPASEEK prediction result for Bacillus subtilis Noc. The red "A"s indicate a putative amphipathic helical region. B Helical wheel projection of the N‐terminus (aa 1-14) showing the presence of hydrophobic (arrow) and polar faces. Residues are coloured according to their properties, greens, hydrophobic; blues, charged; orange, polar, uncharged; and yellow, glycine. The figure was prepared using the tool available at http://rzlab.ucr.edu/scripts/wheel/wheel.cgi. C-L Effects of N‐terminal substitutions on Noc localisation, in strains: DWA211 (F5E), 318 (F9E), 316 (K2E), 212 (R7E), 322 (F5A), 323 (F8A), 325 (F9A), 206 (WT), 328 (S4A) and 329 (S4L), as indicated. Insets show the corresponding phase contrast light microscopy images. Scale bar, 5 μm.
Impaired coolness‐evoked ultrasonic vocalization (USV) in GC‐G‐KO mice A Reduced responsive rate of coolness‐stimulated USV in GC‐G‐KO pups.Figure 7A Reduced responsive rate of coolness‐stimulated USV in GC‐G‐KO pups. Pups were removed from their littermates and dams at postnatal (P) day 2, 4, and 6 and exposed to 15°C. USV was measured by use of the Anabat SD1 Bat detector. The number of responsive pups versus total number of pups tested for each WT and GC‐G‐KO group at different ages is indicated on the top of each bar. *P 0.05.B, C Total number of USV calls (B) and response latency (C) in GC‐G‐KO and WT pups at P2. The horizontal bars indicate the mean. *P 0.05; **P 0.01.
Both CENP-CCR and CENP-Cmotif bind specifically to the CENP-A nucleosome and CENP-CCR easily competes out CENP-Cmotif bound to CENP-A.Figure 2A. Schematic diagram of the full-length CENP-C protein, indicating parts involved in interactions with other proteins or homo-dimerization. Constructs used in this study are depicted below the diagram. B. Native PAGE gel stained with Commassie blue showing complexes formed between CENP-A or H3 nucleosome and CENP-CCR. Lane 1: CENP-A nucleosome, Lanes 2-4: Increasing amounts of CENP-CCR are added to CENP-A nucleosome. Generation of a sharp band with slower mobility indicates formation of a specific CENP-A/CENP-CCR complex. Lane 5: H3 nucleosome. Lanes 6-8: Increasing amounts of CENP-CCR are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-CCR complexes. C. Same experiment as in (B) using CENP-Cmotif. Lane 1. CENP-A nucleosome. Lanes 2-4. Increasing amounts of CENP-Cmotif are added to CENP-A nucleosome. Upon binding CENP-Cmotif, CENP-A nucleosome migrates slower through the gel. Note only modest change in mobility due to small size of CENP-Cmotif, comparing to CENP-CCR in (B). Lane 5. H3 nucleosome. Lanes 6-8. Increasing amounts of CENP-Cmotif are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-Cmotif complexes. D. Native gel showing CENP-CCR competing out CENP-Cmotif bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome, Lane 2: CENP-A/CENP-Cmotif complex. Lane 3-6: Increasing amounts of CENP-CCR are added to the pre-formed CENP-A/CENP-Cmotif complex. Formation of slower migrating bands indicates that longer CENP-CCR is replacing shorter CENP-Cmotif bound to the CENP-A nucleosome. E. Native gel showing the inability of CENP-Cmotif to compete out CENP-CCR bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome. Lane 2 and 6: CENP-CCR/CENP-A nucleosome complex. Lanes 3-5: Increasing amounts of CENP-Cmotif are added to the pre-formed CENP-A/CENP-CCR complex. Formation of smear at high amounts of CENP-Cmotif added, indicates that CENP-Cmotif, at high concentrations, non-specifically binds CENP-A/CENP-CCR complex rather than replacing bound CENP-CCR. Lane 7-9: Increasing amounts of CENP-CCR-short are added to the pre-formed CENP-A/CENP-CCR complex. Formation of bands with higher mobility indicates that smaller CENP-CCR-short is effectively replacing bigger CENP-CCR bound to CENP-A nucleosome.
Figure 5A. Histograms showing the mean distance (megabases, Mb) of observed double crossovers (DCO, red vertical line), compared to 2,000 matched sets of randomly generated distances (RAN, black). The mean distance of the random sets is shown in by the vertical grey line. B. SNPs/kb values were calculated in physical windows of increasing size (kb) around crossover midpoints in wild type (red) and msh2 (blue). The same values were calculated for matched randomly chosen positions (grey). Printed above the plot for each window are circles that are coloured green if crossover SNPs/kb values are significantly different to random (P<0.05), or red if not (P>0.05) (Bonferroni adjusted t-tests). The population analysed (Col×Ct, Col×Ler, Col×CLC) and its genome average SNP frequency (SNPs/kb) is printed above the plots and the number of crossover positions analysed is printed inset. Beneath are plots showing the significance of SNPs/kb differences around crossovers between the indicated genotypes.
PI3Kα inhibition is effective on pro-tumoural features regardless of the genetic landscape of pancreatic adenocarcinoma.Figure 4A, Migration inhibition capacities of PI3K inhibitors at 1 µM were tested on pancreatic tumour cells (R211, PDAC8661, PANC-1, 10593, 10158, R6344, R6430, R6065, R6141). The mean of these values was plotted against the in vitro IC50 for each class I PI3K and each inhibitor. IC50 is determined on recombinant proteins. Pearson correlation tests were performed and its p-values were presented separately for each class I PI3K isoform. isoform. B, individual p-values of these correlation tests were represented for each of the 9 pancreatic (R211, PDAC8661, PANC-1, 10593, 10158, R6344, R6430, R6065, R6141,) breast (MDA-MB-231, MDA-MB-468) and prostate (PC3) cancer cell lines; Pearson correlation p values for each PI3K isoform are plotted separately. n = 3 in each group. C, Cytotoxic capacities of PI3K inhibitors at 10 µM were tested on the 10 pancreatic (R211, PDAC8661, DT4994, PANC-1, 10593, 10158, R6344, R6430, R6065, R6141), breast (MDA-MB-231, MDA-MB-468), prostate (PC3) and acute myeloid cells (NOMO-1, HL-60) and the correlation with the in vitro IC50 for each class I PI3K of each inhibitor was determined. Pearson correlation tests were performed, and p-values represented for each isoform and cell line. n ≥ 3 in each group.
Figure 2Immunoblotting analysis of BCL9 and cell cycle protein expression in double thymidine synchronised cells; the black arrow indicates the delayed band of mitotic BCL9. Immunofluorescence confocal analysis of BCL9 localization on the spindle by antibody staining. Yellow arrows indicate the spindle pole. Scale bars represent: 2 μm for interphase and anaphase; 5 μm for prophase, metaphase and telophase. Immunofluorescence confocal analysis of BCL9 localization on the centrosome. Yellow arrows indicate the centrosome. Scale bars represent: 5 μm for control treatment, 10 μm for BCL9 peptide treatment. Confocal microscope 3D-analysis and radial histograms of the spindle angle in the cells with siRNA treatment; yellow arrows indicate the centrosome. Scale bars represent 5 μm. Confocal (left panel) and live cell imaging (right panel) analysis of chromosome segregation in H2BGFP-cells following siRNA treatment; yellow arrows indicate the typical abnormal chromosomes by BCL9 knockdown. 23 H2BGFP-mitotic cells were monitored for each time experiment; data are shown from three independent experiments. Scale bars represent 5 μm. Quantification of aberrant chromosome segregation rate by live cell imaging of H2BGFP cells upon Wnt3a or DKK1 treatment. 23 H2BGFP-mitotic cells were monitored for each time experiment; data are shown from three independent experiments. Quantification of the duration of NEB to ANA (NEB: nuclear envelope breakdown, ANA: anaphase), NEB to cell death in interphase after cell division (blue colour bar) or in mitosis (dark red bar), or growth well in 30 hours (pink bar) by live cell imaging analysis in monitored H2BGFP cells with different treatments.
Figure 6(A) An alignment of predicted NLxxxL motifs from Far1, Lif1, Slx4, Fin1 and Spc110. In case of Fin1 and Spc110, the motif overlaps with an RxL motif. (B) Autoradiograph showing the cyclin specificity and docking dependency of Lif1 and Slx4 phosphorylation. The experiments were repeated twice, representative examples are shown. (C) NLxxxL is necessary for efficient phosphorylation of Lif1 and Slx4 by Clb5-Cdk1, as shown by the in vitro kinase assay. The experiments were repeated twice, representative examples are shown. (D) Autoradiograph of Clb5-Cdk1-mediated phosphorylation of indicated Fin1 mutants. The experiments were repeated twice, representative examples are shown. (E) Alignment showing the introduction of RxL motif determinants to Far1 NLxxxL motif in mutants analyzed in time-lapse microscopy in panels 'G-H'. (F) Scheme of Far1(1-150 SP)-GFP sensors used for time-lapse microscopy-based analysis of different docking motif variants. All TP sites have been mutated to SP sites. (G) The effect of different docking motifs on the degradation of Far1(1-150 SP)-GFP, where all TP sites are mutated to SP. Plot shows mean±SEM of fluorescence intensities in the cell cycle. (H) The time from Start to the degradation of 50% of Far1(1-150 SP)-GFP with the indicated docking motif was measured in single cells. The numbers above the plot show median values for the sensors, the black lines show median±95% CI. **** and ns denote p-value <0.0001 and >0.05, respectively, for pair-wise comparisons with SP WT using Mann-Whitney U test.
a, TEM (bottom; scale bar, 5 µm) and fluorescence (top; scale bar, 10 µm) of NRK-LC3 cells starved for times shown with enlargements (boxes).figf3a, TEM (bottom; scale bar, 5 µm) and fluorescence (top; scale bar, 10 µm) of NRK-LC3 cells starved for times shown with enlargements (boxes). b, d, Immunoblots of cells shown in a with indicated antibodies (d, after transfection with non-specific (NS) or atg5 RNAi). c, Autophagy (black lines  show  independent experiments) and phospho-S6K percentage (densitometry of data shown in b, red line). e-g, LAMP1-YFP NRK-LC3 cells starved for 2 h and then treated with 100 nM rapamycin for another 4 h (total, 6 h) or 6 h (total, 8 h) analysed by blotting (e) as in b or microscopy (f; scale bar, 5 µm) with quantification (g). Error bars, s.e.m.; n = 3.
plays an essential function in HKC and SCC cells proliferation.Figure 6(A) Colony forming assays of multiple HKC strains from donors of White (213W) and Black (246B, 261B, 285B) origin, which were infected with two HSD17B7 silencing lentiviruses (shHSD2, shHSD4) versus vector control (shCtrl). Gene silencing efficiency is shown in Appendix Fig S6. Cells were plated in triplicate dishes at limited density (1000 cells per 60mm dish) at 5 days after infection and fixed a week later for determination of colony formation as in Fig. 5A. Shown are representative dishes together with data quantification, expressed as numbers of colonies +/- SD. n(dishes per condition)= 3 **p<0.01. 2-tailed unpaired t-test. (B) Sphere forming assays of multiple HKC strains plus/minus HSD17B7 silencing as in the previous panel. Cells were cultured in Matrigel in triplicate dishes, utilizing the same conditions as for the experiments of Fig 2F. Data are expressed as ratio of large spheres (> 2000 pixels>=0.0095mm2) versus total number of spheres (>100 pixels>=0.00047mm2). +/- SD. n(dishes per condition)= 3 **p<0.01; ****p<0.0001, 2-tailed unpaired t-test. (C) EdU labeling assays of multiple HKC strains plus/minus HSD17B7 silencing as in the previous panels. Cells were plated in triplicate dishes 5 days after lentiviral vector infection and selection and pulse labeled with EdU for 3 hrs. Shown are % of EdU positive cells +/- SD. n(dishes per condition)= 3. *p<0.05, **p<0.01; ***p<0.0005; p****<0.0001, 2-tailed unpaired t-test. (D) Colony forming assays of the indicated SCC cell lines plus/minus HSD17B7 silencing as in the previous panels. Cells plated in triplicate dishes 5 days after lentiviral vector infection and selection were tested for clonogenic capability as in (A). Data are represented as number of colonies +/- SD. n(dishes per condition)= 3 *p<0.05, **p<0.01; p****<0.0001, 2-tailed unpaired t-test. (E) EdU labeling assays of the indicated SCC cell lines plus/minus HSD17B7 silencing . Experimental conditions were as in (C). n(dishes per condition)= 3. ****p<0.0001, 2-tailed unpaired t-test. Data are represented as % of EdU positive cells +/- SD. (F) Colony forming assays of SCC13 cells plus/minus lentivirally-mediated HSD17B7 overexpression and subsequent infection with two shRNA silencing vectors as indicated. Experimental conditions were as in (D). n(dishes per condition)= 3 *p<0.05, **p<0.01; ***p<0.0005, 2-tailed unpaired t-test. Data are represented as number of colonies +/- SD.
Figure 4 A-C Exponentially growing cell cultures were arrested in G1 with α-factor (αf) and released in YEPD supplemented with CPT (50 μM). (A) Western blot analysis with anti-Rad53 antibodies  A-C Exponentially growing cell cultures were arrested in G1 with α-factor (αf) and released in YEPD supplemented with CPT (50 μM) (B) Quantitative analysis of Rad53 phosphorylation was performed by calculating the ratio of band intensities for slowly-migrating bands to the total amount of protein. Plotted values are the mean values with error bars denoting SD (n = 3). Statistical analysis: Student"s t-test.; ns, not significant; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001  A-C Exponentially growing cell cultures were arrested in G1 with α-factor (αf) and released in YEPD supplemented with CPT (50 μM) (C) Nuclear division determined by florescence microscopy  D Exponentially growing cell cultures were serially diluted (1:10) before being spotted out onto YEPD plates with or without CPT at the indicated concentrations  E Cells were distributed on YEPD plates with or without CPT at different concentrations to determine colony-forming units after 3 days at 25°C. Plotted values are the mean values with error bars denoting SD (n = 3)
Depletion of ORP5 affects mitochondria morphology and function.Figure 4(A) ORP5, ORP8 and actin levels of HRP-KDEL-expressing HeLa cells treated with Ctrl siRNAs (siCtrl) or siRNAs against ORP5 (siORP5) or ORP8 (siORP8). (B) Electron microscopy of HRP-KDEL-expressing HeLa cells treated with Ctrl siRNAs or siRNAs against ORP5 or ORP8. Red arrows indicate ER-PM contact sites. Scale bar, 2 μm. Insets show representative mitochondria and red arrows in the insets indicate ER-mitochondria contacts. Inset scale bar, 500nm. (C) Representative EM micrographs showing the morphology of mitochondria in siCtrl, siORP5 and siORP8-treated cells. Scale bar, 1 μm. (D) Quantifications of the number of mitochondria with altered morphology in the indicated siRNA conditions. % altered mitochondria ±SEM, n=25 cells and 632-710 mitochondria. *P<0,001 compared to siCtrl. (E) Mitochondrial oxygen consumption rate (OCR) measured in Ctrl and ORP5 siRNA transfected cells. OCR trace was obtained by sequential measurement of basal OCR (OCRBAS), OCR after the addition of oligomycin (OM) and OCR after addition of antimycin A (AA). Note the reduced basal OCR (OCRBAS) compared to ctrl siRNA cells. Error bars denote ±SEM. Data are the mean of 4 independent repeats (n=4), *P<0,05.
LTN1 deletion rescues the ribophagy defect of UBP3-null cells.Figure 1.(A-D) Cells expressing Rpl5-GFP (A and B) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (A and C, left) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts. (right) The ratio between cleaved GFP and full-length protein was quantified for every time point in four independent experiments. a.u., arbitrary unit. (A-D) Cells expressing Rpl5-GFP (A and B) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (B and D) Cells before starvation or starved for 24 h (starvation) were examined both by fluorescence microscopy and differential interferential contrast (DIC). Note that 100% of wild-type (WT) and mutant cells expressed GFP-tagged proteins in the cytoplasm before starvation. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole. (A-D) Cells expressing Rpl25-GFP (C and D) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (A and C, left) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts. (right) The ratio between cleaved GFP and full-length protein was quantified for every time point in four independent experiments. a.u., arbitrary unit. (A-D) Cells expressing Rpl25-GFP (C and D) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (B and D) Cells before starvation or starved for 24 h (starvation) were examined both by fluorescence microscopy and differential interferential contrast (DIC). Note that 100% of wild-type (WT) and mutant cells expressed GFP-tagged proteins in the cytoplasm before starvation. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole.
Concavin-GFP localizes to the periphery of ookinetes and sporozoites A.Figure 2A. Oocyst development of concavin(-) parasites complemented with either the P. berghei gene or the P. falciparum orthologue fused to GFP. Data points represent individual midguts observed between d12-17 post infection of 3 independent cage feeds. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test. B. Average speed of salivary gland sporozoites. Data points represent individual sporozoites of 3 independent biological replicates. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test. C. Sum table of mosquito infections of wild type, concavin(-) and complemented lines expressing either P. berghei (Pb) or P. falciparum (Pf) concavin-GFP. Numbers determined on d17 post mosquito infection. Infection rate contains 3 different countings of at least 20 mosquitoes from different mosquito infections. Note the difference of normally shaped and total sporozoites for concavin(-) parasites. Images show infection of salivary glands. Scale bar: 100 µm D. Pb concavin-GFP and Pf concavin-GFP localization in blood and mosquito stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm. E. Localization of P. berghei concavin-GFP in liver stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm. F. Immunofluorescence images of permeabilized and unpermeabilized concavin-GFP expressing salivary gland sporozoites stained with an anti-GFP antibody. Note that a GFP signal could only be detected after permeabilization, excluding concavin localization on the parasite surface as illustrated in the model. Scale bar: 5 µm.
DMVs are sites of vRNA synthesis.Fig 4.(A) Overview of an infected cell in which regions with different virus-induced modifications are annotated in yellow (DMVs), blue (CM) and orange (DMSs). Several densely-labelled regions containing DMVs, but not the other virus-induced structures are apparent. A close-up of one of these regions (boxed area) is shown in (B). (C, D) Distribution of the autoradiography signal around DMVs (nDMVs = 36, see Material and Methods for selection criteria and details). The data is plotted (C) as a histogram, or (D) normalized by the radius to the DMV centre to account for the increase in the screened area. Scale bars, (A) 5 μm, (B) 500 nm.
Figure 6(A) A collection of serum samples from volunteers working at the CBMSO were taken in the months of June and October 2020 and tested by flow cytometry using Jurkat-S cells at the indicated dilutions. Flow cytometry data are shown as the S/EGFR MFI ratio. Red arrows indicate a loss in titer of antibodies between June and October whereas blue arrows indicate an increase in antibody titer.