EMBO/sd-character-level-ner huggingface dataset:
Phalloidin bound F-actin structure resembles ADP-actin state.Figure 1A. Surface representation of F-actin ADP model, five monomers marked as n series from pointed to barbed end. A phalloidin molecule (yellow stick representation) bound between three actin monomers is highlighted. B. Closer view of phalloidin binding pocket as marked with red box in A. The density of phalloidin from EM map is shown around the ligand. C. Comparison of phalloidin binding pocket residues between apo (in gray) and phalloidin bound (actin monomer colors as indicated in panel A) Key residues with their side chains and phalloidin are represented in stick representation. D. Overlay of F-actin ADP (gray), ADP/Phalloidin (blue) and ADP/Jasplakinolide (orange) shows the D-loop conformations across different structures as indicated.
Figure 8A. Plot of the percentage of nup116Δ cells incubated for 3 h at 37°C with nuclear rim-like accumulations of Chm7-GFP. Error bars are SD from the mean from 3 independent replicates of >100 cells. p values from unpaired student's T-test where *** is p ≤ 0.001.B. Deconvolved inverted fluorescence micrographs of Chm7-GFP in nup116Δ cells at either 23°C or grown for 3 h at 37°C. Percentages ± SD of the proportion of cells with Chm7-GFP NE foci are shown under each panel (see Figure EV5F). C. Electron micrographs showing herniations of the NE in nup116Δ cells after incubation for 3 h at 37°C. N denotes nuclear interior. Scale bars are 100 nm. D. 10-fold serial dilutions of indicated strains grown in medium permissive (-URA) or restrictive (5-FOA) of the retention of a URA3/NUP116 plasmid. Images acquired after incubation for 3 days (left) or 6 days (right) at 23°C.
Protozoan and human tubulin and microtubule structures.Figure 1(A) Coomassie stained SDS-PAGE showing purified T. thermophila tubulin. (B) A representative cryo-electron micrograph of paclitaxel-stabilized T. thermophila MTs. (C) Kymographs obtained by TIRF microscopy showing the dynamics of T. thermophila MTs at the indicated concentrations in µM. (D) Plot of T. thermophila MT plus-end growth rates as a function of tubulin concentration. Error bars represent the mean +/- 95% CI. For each concentration, data is obtained from at least 2 independent experiments (n=41). (E) Cryo-electron microscopy density map of a 14 protofilament, paclitaxel-stabilized T. thermophila MT with pseudo helical symmetry applied. Left: outside view. The α- and β-tubulin monomers are shown in surface representation and colored in dark and light gray, respectively. Red arrow shows the location of the seam. Right: tubulin dimer models fitted into a sliced, transparent and zoomed-in view. The paclitaxel structure in stick representation is highlighted in magenta. Lateral contacts between protofilaments are indicated in blue (H2-S3 in α2 and β2), red (H1-S2 in α2 and β2) and green (M loop in α1 and β1). (F) Cartoon representation of the crystal structures of T. thermophila tubulin-D1 (left) and recombinant H. sapiens tubulin-D1 (right) complexes. Bound nucleotides are displayed in stick representation, the
Class 1 sites are in black font.f2Class 1 sites are in black font. Additional sites found in Class 1 proteins are in red (HCT116PARKIN) or blue font (HeLaPARKIN). Site overlap in HCT116 and/or SH-SY5Y: magenta, orange, and green octagons. Dotted lines: interacting proteins. Rectangles represent Class 2 substrates. Red or blue boxes refer to additional sites identified in either HCT116PARKIN and HeLaPARKIN cells (Supplementary Table 2). MIM, mitochondrial inner membrane. * and ∧, protein levels decrease or increase, respectively, upon depolarization.
Ectopic expression of Atg14L in the plasma membrane.Figure 3.(a) A549 cells were transiently transfected with adenovirus expressing GFP-Atg14L or GFP-Atg14LCAAX and subjected to starvation. The cells were fixed and stained with anti-Beclin-1 antibody. Higher magnification of the boxed areas is shown below. (b) HEK293 cells were cotransfected with MEF-hVps34 and GFP or GFP-Atg14LCAAX and stained with anti-Flag antibody. Bars, 10 µm.
Figure 8(A) qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from the lungs of WT or Galectin-9 KO mice at 3 days post intraperitoneal administration of AG (100 μg). Data information: Data are means + SD of the indicated number of mice from 1 of n=3 independent experiments and each symbol represents data from 1 mouse. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001. Scale bar, 200 μm. (B, WT or Galectin-9 KO mice were intraperitoneally treated with AG for 3 d in the absence or presence of the MMP inhibitor marimastat (10 mg/kg) given intraperitoneally prior to AG stimulation. Lung sections stained with H&E Data information: Data are representative of n=3 independent experiments. C) quantification of lung lesion burden from H&E-stained sections Data information: Data are means + SD of the indicated number of mice from 1 of n=3 independent experiments and each symbol represents data from 1 mouse. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001. Scale bar, 200 μm. (D, WT or Galectin-9 KO mice were intranasally infected with H37Rv for 4 weeks in absence or presence of intranasally administrated AG aptamers (1 μg) once at a 1 week-interval. Lung sections stained with H&E Data information: Data are representative of n=3 independent experiments. E) quantification of lung lesion burden from H&E-stained sections Data information: Data are means + SD of the indicated number of mice from 1 of n=3 independent experiments and each symbol represents data from 1 mouse. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001. Scale bar, 200 μm. (F) CFU quantification of the bacterial titers of lung tissue homogenates from WT or Galectin-9 KO mice intranasally infected with H37Rv for 4 weeks in absence or presence of intranasally administrated AG aptamers (1 μg) once at a 1 week-interval. Data information: Data are means + SD of the indicated number of mice from 1 of n=3 independent experiments and each symbol represents data from 1 mouse. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001. Scale bar, 200 μm.
Figure 2(A) Flow cytometry dose response curve for cell binding of 4D9 mAb (EC50= 0.29nM), 4D9 Fab (EC50=0.17nM), and isotype to HEK cells stably overexpressing mouse TREM2. Data represent the mean ± SEM (n=2). (B) In vitro ADAM17 sheddase activity is blocked by 4D9-effectorless mAb and 4D9 Fab fragment but not an isotype control. Fluorescence polarization of FAM conjugated TREM2 stalk peptide was detected in the presence or absence of ADAM17 and 4D9 mAb, 4D9 Fab and isotype control. Data represent the mean ± SEM (n=6). One-way ANOVA, Tukey's post hoc test; p (4D9 Fab vs 4D9 mAb) = 0.8855; p (4D9 Fab vs uncleaved) < 0.0001; p (4D9 mAb vs uncleaved) < 0.0001; n.s., not significant. (C) ELISA-mediated quantification of sTREM2 in conditioned media from  (D) AlphaLISA-mediated quantification of pSYK levels in HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12 treated with a dose titration of 4D9 mAb, 4D9 Fab or isotype for 5 minutes. Data represent the mean ± SEM (n=3). (E) AlphaLISA-mediated quantification of pSYK levels in HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12 or empty vector treated with 1mg/ml POPC/POPS liposomes and 20µg/ml 4D9 mAb, 4D9 Fab or isotype for 5 minutes. pSYK levels were also determined for cells treated with liposomes only. Data represent the mean ± SEM (n=3). Two-way ANOVA, Tukey's post hoc test (cell line effect: F1,16=365.7, p=<0.0001; treatment effect: F3,16=39.35, p<0.0001; cell line x treatment effect: F3,16=38.75, p<0.0001); p (no Ab vs isotype) = 0.6218; p (no Ab vs 4D9 mAb) < 0.0001; p (no Ab vs 4D9 Fab) = 0.7301; p (isotype vs 4D9 mAb) < 0.0001; p (isotype vs 4D9 Fab) > 0.9999; p (4D9 mAb vs 4D9 Fab) < 0.0001; n.s., not significant. (F) Schematic representation of the proposed mechanism of action of antibody 4D9. Binding of 4D9 to TREM2 leads to receptor clustering on the cell surface thereby driving downstream pSYK signaling. At the same time, cell surface levels are enhanced by inhibition of ectodomain shedding, potentially because of the inability of proteases to cleave dimeric substrates.
MiniSOG‐tagged Mpl reveals that Mpl enters the autophagic pathway from specialized ER exit sites.Figure 6Confocal fluorescent images obtained after transient expression of Mpl tagged with mKO2 and miniSOG in K562 (A) and HEL (B) cells. Electron micrographs of K562 (C) and HEL (D) cells transiently expressing MplmKO2‐miniSOG. Dark precipitate indicates the presence of the miniSOG‐tagged Mpl protein after photo‐precipitation of DAB. White boxes indicate original location of higher magnification images with corresponding letters. E-G) High magnification electron micrograph of K562 cells treated in the same conditions as Figure C, showing MplmKO2‐miniSOG dark precipitate into autophagic carrier vesicles (black arrows, Figure E). Osmiophilic reaction products also heavily stain mature AP in these preparations (black arrows, Figure G). H) High magnification electron micrograph of HEL cells treated in the same conditions as Figure D, cells transiently expressing MplmKO2-miniSOG , showing different stage of formation of APs (black arrows numbered from stages 1-4). Scale bars = 5 µm (A-D) or 100 nm (E-H).
Figure 6(A) Schematic showing experimental setup to measure rates endocytosis in BV-2 cells. Control (MIP empty vector) cells were plated together with cells stably expressing different Siglec constructs. Cells are allowed to adapt to the same media environment for 24 hours, then fluorophores are added to media. Cells with endocytosis substrates (Aβ, Dextran) were assayed after 3 hours, while phagocytosis substrates (FluoSpheres) were assayed after 24 hours. Populations are separated and relative uptake is measured using flow cytometry. (B-C) Flow cytometry quantification of fluorescent monomeric Aβ uptake and Siglec-F expression for (B) Siglec-F and (C) Siglec-F 2xY->F. Siglec-expressing cells were plated with empty vector control cells in the same well alongside fluorescent substrates. Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of monomeric Aβ, Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group. Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of 10,000 MW Dextran Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group. Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of 1 μm FluoSpheres. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group. (G) pHrodo Dextran uptake in BV-2 cells with stable expression of Siglec-F estimated by Incucyte measurements. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. (H) pHrodo dextran uptake in BV-2 cells stably expressing Siglec-F with 0 - 2.5 μM SHP099 treatment. Data is from the same experiment as (G). (I) Confluency doubling times for BV-2 cells with stable expression of Siglec-F with 0 - 2.5 μM SHP099 treatment. Data is from the same experiment as (G). Bars indicate mean ± 95% CI; n = 4 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated 2xY->F group.
a) Atg8 (LC3) localizes to the MR during abscission.figf2(a) Atg8 (LC3) localizes to the MR during abscission. The upper panels show stills from time-lapse microscopy of GFP-Atg8-expressing cells. The arrowhead points to the accumulation of Atg8 at the site of the MR during abscission. The enlarged clipping on the right shows the midbody area at time-point 68 min. Scale bar is 10 μm. (b) Cytosolic MRs are found inside Atg8-positive vesicles. U2OS cells stably expressing GFP-Atg8 were fixed and stained for MKLP1 (red) and α-tubulin (blue). Scale bars are 10 μm and 1 μm for the enlarged boxed areas. Putative different stages of autophagosomes (left) and lysosomes (right) are shown. (c) Juxtanuclear MRs are found in Rab7- and LAMP2-positive lysosomes. U2OS cells stably expressing DsRed-Rab7 were fixed and stained for MKLP1 α-tubulin and DAPI (left panels); wild-type U2OS were stained for MKLP1 and LAMP2 (right panel). Scale bars are 10 μm. The insets are enlargements of the engulfed MRs. Scale bars are 1 μm.
a) Representative in situ hybridization of frozen sections obtained from colon biopsies of genotyped healthy controls (n = 40) or individuals with Crohn's disease (CD) (n = 67) without or with active inflammation and labeled for miR-196A.figf2(a) Representative in situ hybridization of frozen sections obtained from colon biopsies of genotyped healthy controls (n = 40) or individuals with Crohn's disease (CD) (n = 67) without or with active inflammation and labeled for miR-196A. (L, lumen; LP, lamina propria). Scale bars in the upper panel, 25 μm; scale bars in the lower panel, 10 μm.  (b) Epithelial or laminal fractions were captured from sections of biopsies of healthy controls (n = 8) or individuals with Crohn's disease with no inflammation (n = 16), quiescent (defined as low-grade inflammation) (n = 8) or acute inflammation (n = 8) using laser capture microdissection. After RNA extraction, miR-196A (black bars) and miR-196B (white bars) relative expression was analyzed using RNU19, 44 and U6. To overcome possible inter-individual bias, the lamina propria fraction value was used for relative quantification. Due to high differences in expression between healthy and inflamed tissues, the results are presented as a log2-fold ratio. Error bars indicate the s.d. of the ΔΔCt values. (c) Representative in situ staining for IRGM of TMAs from colon biopsies of healthy individuals or individuals with Crohn's disease with a defined genotype in a healthy non-inflamed or an acute inflamed phase. Scale bars, 32 μm. (d) Mean (black line), s.e.m. (white box) and 95% CI of the mean of the IRGM expression level for 40 healthy subjects (32 individuals with C/C and 8 individuals with C/T) and 67 individuals with Crohn's disease (45 with C/C and 22 with C/T) with quiescent or inflamed colon mucosa. We performed statistical analysis using ANOVA (P = 0.0015) and an unpaired Student's t-test (the one tail P value is indicated on the figure).
DGAT1 activation drives lipid droplet biogenesis upon iron depletion.Figure 4a. Line diagram of temporal lipidomics workflow in human ARPE-19 cells. b. Multivariate modelling of the lipidome in cells treated with DFP over time. Sparse PLS-DA (sPLS-DA) was performed using Metaboanalyst from LC-MS datasets from three independent biological experiments each with four technical replicates per time point in each experiment. c. Lipidomic analysis. Hierarchical clustering heatmap analysis showing alterations in lipid species in ARPE19 cells treated with DFP for the length of time indicated. Each coloured cell on the map corresponds to a z-score value. The hierarchical clustering method of one minus Pearson correlation was applied (n=3, with 4 technical replicates per biological replicate). d. DFP-induced Lipid droplet accumulation is dependent on DGAT1. Representative photomicrographs of ARPE19 cells treated with DFP (1mM) for 7 h followed by treatment with LD inhibitors (5 μM) as indicated. Cells were fixed after 24 h of DFP treatment. Neutral lipid droplets were stained by BODIPY493/503 and imaged by confocal microscopy after 24 h of DFP treatment ('i' denotes the presence of inhibitor e.g. DGAT1i = DGAT1-inhibition). Scale bar = 5 μm. e. Quantitation of lipid droplets area per cell from experiments in 2d. Data are represented as mean ± SEM (n=3 with at least 80 cells analysed per condition in each biological replicate). f. Acute inhibition of LD biosynthesis by DGAT1. Quantitation of mean LD numbers per ± SEM (n=2, with 3 technical replicates per biological experiment. Between 1000-2000 cells analysed per condition). g. DGAT1 regulates triglyceride formation upon iron chelation. Hierarchical clustering heatmap analysis showing alterations in triglyceride lipid species in ARPE19 cells treated with DFP for 24 h and lipid droplet inhibitors (5 μM) for 17h as indicated (i denotes the presence of inhibitor e.g. DGAT1i = DGAT1-inhibition). Each coloured cell on the map corresponds to a z-score value. The hierarchical clustering method of one minus Pearson correlation was applied and statistical significance was determined by a T-test (n=3, with 5 technical replicates per biological replicate).
Figure 3(A) CRISPR/nCas9-mediated ablation of Afadin protein levels in a clonal cell population termed Afdn-KO. (B) Oil red O staining and gene expression of selected adipogenic genes in WT-1 and Afdn-KO differentiated adipocytes (n=3). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance. (C) Wild-type, Afdn-KO and Afdn-KO with re-expression FLAG-tagged WT I-Afadin or a non-phosphorylable form (S1795A) stimulated with vehicle or insulin (n=4) for Immunoblotting. (D) Quantification of insulin-induced Akt phosphorylation from immunoblots. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance. (E) Real-time measurement and (F) quantification of insulin-induced fatty-acid uptake (n=4). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.
Comparison with DNA methylation mutants and meiocytes.Figure 8(a) DNA methylation differences between stem and non-stem cells and their enrichment within DMRs of different epigenetic mutants using permutation tests (data from Stroud, Greenberg, Feng, Bernatavichute and Jacobsen 2013). Mutants are in alphabetical order according to the gene acronym. Red indicates an enrichment of methylated DNA in the respective context and blue a depletion. (b) Principal component analysis for relatedness between CHH DMRs at TEs in meiocytes (data from Walker et al. 2018) and stem (+) and non-stem (-) cell nuclei of 7, 14, or 35 day-old plants (D7/14/35).
Figure 1(A) An illustration on the definition of focal, arm, and chromosome level somatic copy-number alterations (SCNAs). (B) Example of somatic copy-number alterations detected in a single TCGA sample (TCGA-02-0003, GBM) with copy number gain in red and copy number loss in blue. The x axis represents the chromosomal location and the y axis shows the log2 fold change in the intensity of the corresponding region relative to diploid. (C) Heatmap showing the average fold change of copy number gain (red) and loss (blue) of chromosomal p and q arm events across 32 tumor types in TCGA. (D) Visualization of the distribution of total SCNA events (loss and gain) by category across tumors grouped by 32 tumor types in TCGA. From top to bottom, plots depict events of chromosomal, arm, and focal level SCNA respectively.
Figure 2TetON-hCDC14A-GFP RPE1 cells were serum starved for 48 h prior to fixation and immunofluorescence microscopy. hCDC14A-GFP colocalizes with Phalloidin 555 conjugated dye that marks F-actin fibers. The boxed area was 10-fold enlarged and displayed below the corresponding images to indicate co-localization of hCDC14A-GFP and F-actin. Line scan inside the magenta colored box showed in (A) Sub-centrosomal localization of hCDC14AC278S-GFP was determined by correlative positioning of the GFP signal with the proteins Arl13B and IFT88 (cilia), C-Nap1/ninein (proximal end), CEP164 (distal appendage) and ODF2/ ninein (subdistal appendage). Results are fitted in the cartoons adjacent to the respective panels for better visualization The cartoon summarizes the localization of hCDC14A on cilia. The distal appendage protein CEP164 marks the mother basal body. The C-Nap1 organized centrosome linker connects the mother with the daughter basal body (black lines). ODF2 and ninein mark the subdistal appendages. Note, ninein has a second localization at the distal end of basal bodies. hCDC14A localized to the subdistal appendages and the C-Nap1 marked proximal end of the basal bodies
TFEBmt reduces lysosomal size, but induces apoptosis in a subset of PD myotubes.Figure 3A. Immunostaining of non-infected cells (day 13 in differentiation medium) and cells infected with a mutant form of TFEB (TFEBmt) with LAMP1 (red) and Flag (green). Ad-TFEBmt was added to the myotubes for 72 h on day 10 in differentiation medium. Ad-TFEBmt-infected cells show massive accumulation of TFEB in the nuclei and significant reduction in lysosomal size, similar to that seen with TFEB. B. Western blot of cell lysates confirms the presence of TFEB in the nuclear fraction; the different intensities of the two bands corresponding to LAMP1 protein in untreated and TFEBmt-treated samples may reflect the differences in the glycosylation pattern. C. Western blot of protein lysates from untreated, Ad-null, TFEB, and TFEBmt-treated myotubes with anti-caspase-3 antibody. No activated (cleaved) products are detected in any condition. α-Tubulin was used as a loading control. D. TUNEL assay shows the presence of some apoptotic cells in Ad-TFEB-infected cultures, but not in control cultures infected with adenovirus alone (n = 3). Bar: 10 µm for all panels.
NMR analysis of activated BakΔTM on lipid nanodiscs.Figure 4(a) Size exclusion chromatogram of nanodiscs (black) and BakΔTM bound to nanodiscs (red). Inset: SDS-PAGE confirms the presence of Bak-loaded nanodiscs. (b) Overlay of 2D-[15N, 1H]-TROSY spectra of BakΔTM with (red) and without (blue) nanodiscs. (c) 2D-[15N, 1H]-TROSY spectrum of BakΔTM in nanodiscs after Bid-BH3 activation with the assigned resonances indicated. (d) {1H}-15N-heteronuclear NOE of BakΔTM in the inactive state (blue) and the active state in nanodiscs after Bid-BH3 activation (green). α-helices in Bak are shown at the top. Residues 25-54 of the activated state are magnified. Chemical shift-derived secondary structure elements are indicated at the bottom. (e) Left: Overlay of 2D-[15N, 1H]-TROSY spectra of BakΔTM in [Ni2+]-caged liposomes (magenta) and Bid-BH3 activated BakΔTM in nanodiscs (green). Right: Plot of the relative NMR intensity of Bid-BH3 activated BakΔTM in nanodiscs. Peaks that are also visible in liposomes are indicated by horizontal lines in magenta at the bottom, while the chemical shift-derived secondary structure elements are indicated at the top.