EMBO/sd-character-level-ner huggingface dataset:
Figure 1A. GFP-Split U2OS-ACE2 were co-cultured at a 1:1 ratio and infected with SARS-CoV-2. Syncytia formation and cell death was monitored by video microscopy or at endpoint using confocal microscopy and high content imaging. B. Still images of GFP (syncytia) and Propidium Iodide (PI) (cell death) at different time-points. Scale bar: 100 µm. C. Quantification of U2OS-ACE2 fusion and death by time-lapse microscopy. Results are mean±sd from 3 fields per condition. D. S staining of infected U2OS-ACE2 cells analyzed by immunofluorescence. The Hoechst dye stains the nuclei. Scale bar: 40 µm. E. Surface S staining of infected U2OS-ACE2 cells analyzed by flow cytometry. Results are representative of at least three independent experiments.
a, DARTS identifies ATP5B as an &agr-KG-binding protein.f2a, DARTS identifies ATP5B as an alpha-KG-binding protein. Red arrowhead, protected band. b, DARTS confirms alpha-KG binding specifically to ATP5B. IB, immunoblot. c, Inhibition of ATP synthase by alpha-KG (released from octyl alpha-KG; Supplementary Notes). This inhibition was reversible (data not shown). d, e, Reduced ATP levels in octyl alpha-KG-treated normal humanfibroblasts (**P = 0.0016, ****P  0.0001; by t-test, two-tailed, two-sample unequal variance) (d) d, e, Reduced ATP levels in octyl alpha-KG-treated worms (day 2, P = 0.969; day 8, *P = 0.012; by t-test, two-tailed, two-sample unequal variance) (e). RLU, relative luminescence units. f, g, Decreased oxygen consumption rate (OCR) in octyl alpha-KG-treated cells (***P = 0.0004, ****P  0.0001; by t-test, two-tailed, two-sample unequal variance) (f) f, g, Decreased oxygen consumption rate (OCR) in octyl alpha-KG-treated worms (P  0.0001; by t-test, two-tailed, two-sample unequal variance) (g). h, alpha-KG, released from octyl alpha-KG (800 µM), decreases state 3, but not state 4o or 3u (P = 0.997), respiration in mitochondria isolated from mouse liver. The respiratory control ratio is decreased in the octyl alpha-KG- (3.1 ± 0.6) versus vehicle-treated mitochondria (5.2 ± 1.0) (*P = 0.015; by t-test, two-tailed, two-sample unequal variance). Oligo, oligomycin; FCCP, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone; AA, antimycin A. i, Eadie-Hofstee plot of steady-state inhibition kinetics of ATP synthase by alpha-KG (produced by in situ hydrolysis of octyl alpha-KG). [S] is the substrate (ADP) concentration, and V is the initial velocity of ATP synthesis in the presence of 200 µM octanol (vehicle control) or octyl alpha-KG. alpha-KG (produced from octyl alpha-KG) decreases the apparent Vmax (53.9 to 26.7) and Km (25.9 to 15.4), by nonlinear regression least-squares fit. c-i, Results were replicated in two independent experiments. Mean ± standard deviation (s.d.) is plotted in all cases.
Figure 6A Bar plot showing phospho-ERK1/2 over total ERK1/2 levels in vehicle- and KA-treated WT (closed and open black circles, vehicle-treated WT n=6; KA-treated WT n=6) and Fxr2 KO mice (closed and open magenta circles, vehicle-treated Fxr2 KO n=5; KA-treated Fxr2 KO n=6). *** p < 0.001 comparison between vehicle- and KA-treated WT (two-way ANOVA: interaction effect between genotype and treatment: F1,19 = 24.66, p < 0.001). Data are presented as mean ± SEM. B Phospho-ERK1/2 levels in vehicle- and pilocarpine-treated WT (n=4-6) and Fxr2 KO mice (n=4-6). Both genotypes responded with long-lasting seizures upon pilocarpine administration * p < 0.05 (two-way ANOVA: main effect for treatment: F1,16 = 10.98, p < 0.01). Data are presented as mean ± SEM. C Phospho-ERK1/2 levels in control and KA-treated WT (both n=6) and Fxr2 KO mice (both n=6) sacrificed at different time intervals [0, 20, 40 and 60 min]. * adjusted p < 0.05, ** adjusted p < 0.01 (two-way ANOVA: main effect for genotype: F1,40 = 5.99, p < 0.05; interaction effect genotype and time; F3,40 = 4.11, p = 0.012; p < 0.05 and p < 0.01 with Bonferroni correction for 40 and 60 min, respectively). Data are presented as mean ± SEM.
AAV-VHH-B9 prevents synaptic impairment in AppNL-G-F mice(A) Examples of the fEPSP traces recorded under baseline conditions (left) or post-high frequency simulation (HFS) (right) in acute hippocampal slices obtained from AppNL-G-F mice injected with either AAV-VHH-B9 (magenta) or AAV-GFP (black).Figure 5(A) Examples of the fEPSP traces recorded under baseline conditions (left) or post-high frequency simulation (HFS) (right) in acute hippocampal slices obtained from AppNL-G-F mice injected with either AAV-VHH-B9 (magenta) or AAV-GFP (black). The break in the traces is due to removal of a stimulus artifact. (B) Time course of averaged fEPSP amplitudes relative to baseline average for each experimental group. 3 periods of HFS were applied at time points indicated with black arrows. Vertical dashed lines define the boundaries of the temporal window used to produce the plots in (C). Results are the average fEPSP amplitude ± SEM determined across all recorded slices (2 biological replicates per mouse, 5 independent mice per experimental group) for each time point measured. (C) Average fEPSP amplitude values at individual acquisition points between min 75 and 90 of the recording (vertical dashed lines in B) across experimental groups. Individual symbols represent the averaged fEPSP amplitude measured in one animal (2 slices), with bars representing average ± SEM across all slices.
Native Mass spectrometry and negative–stain EM of the yNap1–H2A–H2B particle.Figure 5A EM analysis of the yNap1c2-H2AΔ14-H2BΔ28 complex. Representative rawimage of negatively-stained particles.B Representative particles (1st row), class averages coming from the projection matching procedure (2nd row) and comparison of the class averages with projections from a final model generated after four projection matching cycles (3rd row). C From left to right: Crystal structure of the (yNap1c2-H2A-H2B)6 complex in two different orientations (brown); 60 Å-filtered structure of the complex (magenta); 3D reconstruction of the complex from negatively stained particles (green) and fit of the crystal structure into the EM map. D-I Mass spectra of yNap1 variants in the absence and presence of H2A-H2B. yNap1 in complex in the absence (D) or presence of H2A-H2B (E). yNap1c in the absence (F) or bound to H2A-H2B (G). The oligomerization mutant yNap1_IF1 alone (H) or in complex with H2A_IF2Δ14-H2BΔ28 (I). yNap1 is shown in green circles and H2A-H2B as red and blue asterisks.
GEMC1 is required for spermatogenesis.Figure 2. Example of dissected testes from littermate mice of the indicated genotypes at 2 months of age. B. Testes from Gemc1-/- mice have strongly reduced cellularity compared to Gemc1+/+ or Gemc1+/- animals, as measured by disaggregation of the tissue and cell counting with a Neubauer Chamber (n=3). Mean and standard deviation are indicated. C. Histological analysis of tubules from 6-week-old Gemc1-/- mice revealed thinning of the spermatogenic cell layer and decreased numbers of elongated spermatids. Scale bars = 100 µm (left panels) and 50 µm (right panels). D. Sperm counts from the cauda epididymis of wild type and Gemc1-/- mice revealed no sperm in mice lacking GEMC1 (n=3). Mean and standard deviation are indicated. E. Examples of sperm morphology from spermatocyte spreads from 2 month old mice. Scale bars = 10 µm. F and G. Histological evaluation of the caput epididymis in sagittal (F) and coronal (G) orientation from 2 and 4 month old animals, respectively. Note the absence of luminal spermatozoa in the Gemc1-/- mice compared to wild type. Scale bars = 100 µm (F, left panels) and 50 µm (F, right panels and G).
Figure 1A) HeLa cells were cultured in complete growth or starvation (STV) media for 30'. Cell lysates were immunoprecipitated (IP) with anti‐TFG antibody or IgG as a negative control. Immunoprecipitated complexes were analysed by WB to detect TFG, ULK1, ATG13, and FIP200 as indicated. B) HeLa cells were cultured in complete growth (FED) or starvation (STV) media for 1h. Cells were fixed, permeabilized, and labelled with anti-TFG (red) and anti-ULK1 (green) antibodies respectively. Hoechst was used to stain nuclei. Co-localization analysis were performed by ImageJ plugin Jacop and reported in the graph (right). Values of Mander's coefficient for ULK1 are expressed as mean ± SEM. Significance was assigned by unpaired Student's t‐test. **, P<0,01. (n = 3 independent experiments, n=12 fields analysed). Scale bar 5 μm. C) HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) for 48h. Protein extracts were analysed by WB to detect ULK1, TFG and ACTIN as indicated. Densitometry analysis of ULK1 over ACTIN is shown (right). All values are mean ± SEM. Statistical analyses were performed by two‐tailed Student's t‐test. ***, P<0.001. (n=3 independent experiments). D) HeLa cells were co-transfected with control siRNA and an empty vector (EV), or 3'UTR TFG siRNA together with an EV or HA-TFG plasmids. Protein extracts were analysed by WB to detect ULK1, TFG and ACTIN as indicated. Densitometry analysis of ULK1 protein levels normalized over ACTIN is also reported (right). All values are expressed as the mean ± SEM. Statistical analyses were performed by one-way ANOVA followed by Tukey's multiple comparison test. *, P<0.05; **, P<0.01. (n=3 independent experiments). E) HeLa cells were transfected as in C), grown in the presence or not of cycloheximide (CHX) for indicated time periods. Protein extracts were analysed by WB to detect ULK1, TFG and ACTIN as indicated. Densitometry analysis of ULK1 normalized over ACTIN is also shown (right). Values are mean ± SEM Statistical analyses were performed by multiple t-test. *, P<0.05. (n=3 independent experiments). F-G) HeLa cells were treated as in B) and then fixed and stained with ULK1 (green) (F) or ATG13 (red) antibodies (G) respectively. Hoechst was used to stain nuclei. Graphs (right) show the average of both ULK1 (F) (n=3 independent experiments, n=11 fields analysed) or ATG13 (n=3 independent experiments, n≥16 fields analysed) (G) puncta number per cell and both ULK1 (n=3 independent experiments) (F) or ATG13 (n=3 independent experiments) (G) fold of increase of the average size respectively. White arrows indicate most representative puncta of analysed conditions. All data are reported as the mean value ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ****, P<0.0001 or by unpaired Student's t‐test when two groups were compared. *, P<0.05; ****, P<0.0001. Scale bar 5 μm.
Figure 4Representative images showing increased mitochondrial association with Rheb in AD axons, relative to that of WT littermate controls. Note that CCCP treatment enhances Rheb localization to mitochondria. The percentages of white arrow-marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP quantitative analysis The percentages of white arrow-marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP were quantified, respectively C- Representative dual-channel kymographs showing defective retrograde transport and mitophagic accumulation within AD axons. mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified quantitative analysis , the numbers of Mito, AVs, and Mito-AVs, and the percentage of mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified, respectively quantitative analysis Relative motility of AVs and Mito-AVs/mitophagosomes in WT or mutant hAPP axons with and without CCCP were quantified F,G Quantitative analysis (F) and representative blots (G) showing that ∆ψm depolarization augments mitophagy activation in cortical neurons cultured from AD mouse brains. Note increased localization of Rheb along with LC3-II and Rab7 to mitochondria in mutant hAPP Tg neurons after CCCP treatment (red box). Data were quantified from three independent repeats. P: post-nuclear supernatants; M: mitochondria-enriched membrane fractions; S: cytosolic supernatants. Quantitative analysis Mean fluorescence intensity ratios evoked by the two excitation wavelengths (405 nm and 488 nm) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT controls representative images showing abnormal accumulation of oxidatively damaged mitochondria in AD axons, which is exacerbated after dissipating ∆ψm. Mean fluorescence intensity ratios evoked by the two excitation wavelengths (405 nm and 488 nm) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT controls
Atg16L1 Mutants Unable to Bind WIPI2 Cannot Rescue LC3 Lipidation or LC3 Recruitment to Salmonella in Atg16L1Δ/Δ MEFs.Figure 7(A) Atg16L1Δ/Δ MEFs were transiently transfected with either mock, FLAG-tagged Atg16L1 WT, ERER, or DRDR. After 2 hr in EBSS, cells were fixed and labeled with anti-LC3 and anti-FLAG antibodies and visualized by confocal microscopy. Scale bars, 20 μm.(B) Statistical analysis of (A) was performed using one-way ANOVA with Dunn's posttest. ∗p < 0.05. SEM for n = 3. (C) Atg16L1Δ/Δ MEFs were transiently transfected with either mock, FLAG-tagged Atg16L1 WT, E226R E230R (ERER), or D237R D239R (DRDR) before analysis by immunoblotting.(D) Statistical analysis of (C) was performed by one-way ANOVA with Tukey's post hoc test. SEM for n = 3. ∗p < 0.05. (E) HEK293A cells were treated with either RISC-free control or WIPI2 siRNA before infection with Salmonella (moi = 100) for 1 hr, labeled with anti-LC3, WIPI2, and p62 antibodies, and followed by confocal analysis. Scale bars, 5 μm.(F) Statistical analysis for (E) was performed using an unpaired Student's t test. ∗p < 0.05. SEM for n = 3. (G) Atg16L1Δ/Δ MEFs were transiently transfected with FLAG-Atg16L1 WT, FLAG-Atg16L1 ERER, or FLAG-Atg16L2 DRDR 24 hr before being infected with Salmonella (moi = 25) for 1 hr, labeled with anti-LC3, anti-p62, and anti-FLAG antibodies, and followed by analysis by confocal microscopy. Scale bars, 5 μm.(H) Statistical analysis of (G) was performed by one-way ANOVA with Dunn's post hoc test. SEM for n = 4. ∗p < 0.05. See also Figure S7.
Figure 6Cut-away views through the structure at positions indicated in the inset schematic end-on views. The T4 proteins are shown in the ribbon diagram representation with the same color scheme Cut-away end-on views (looking from the capsid) at positions indicated with the red lines
XIAP-RIPK2-TAB1 axis supports neutrophil infiltration and melanoma growth in B16 mouse melanoma A Intensities of CD177 and XIAP immunostainings in tumour sections from 55 patients were visually scored.Figure 4A Intensities of CD177 and XIAP immunostainings in tumour sections from 55 patients were visually scored. The numbers of melanomas with different intensities were plotted into the table as a percentage of the total. Specific staining intensity for XIAP and CD177, corresponding to relative amounts of infiltrated neutrophils, was arbitrarily set as the following: -, not expressed; +, low; ++, moderate; and +++, strong expression. Representative IHC pictures (right panel). B Representative IF (neutrophils staining) in B16F1 WT, XIAPKO, TAB1KO or RIPK2KO tumours (Scale bar 50 µm) and quantification of Ly6G+ cells. The sum of Ly6G-positive cells from 5 randomly selected areas of the tumour (5 mice per genotype) was represented. Dots represent individual mice (n=5). C B16F1 WT, XIAPKO (clone 2) subcutaneous melanoma tumour growth in BL/6 WT mice (n=5). Mice were intra-peritoneal injected with IgG antibody as a negative control or Ly6G antibody 1 day prior to the subcutaneous injection of the B16F1 WT or XIAPKO cells. D Representative IF (neutrophils) scale bar 50 µm and quantification of Ly6G+ cells in tumours from C. The sum of Ly6G-positive cells from 5 randomly selected areas of the tumour (4 mice per genotype) was represented. Dots represent individual mice (n=4).
Figure 5A. Triple knock-in (3KI) mESC line that simultaneously reports on ectoderm fate, (marked by the expression of Sox1::GFP), mesendoderm fate (marked by the expression of T::3xTagBFP, T is also known as Brachyury), or definitive endoderm fate (marked by the expression of Eomes::mCherry). B. Ectoderm differentiation of the 3KI mESC line (marked by the expression of Sox1::GFP). C. Mesendoderm differentiation of the 3KI mESC line (marked by the expression of T::3xTagBFP). D. Endoderm differentiation of the 3KI mESC line (marked by the expression of Eomes::mCherry). E, F. Spontaneous differentiation of the 3KI mESC line leading to the coexistence of cells belonging to the three germ layers. Bar: 100 μm.
The DNA damage response at telomeres uncapped through TRF2 depletion does not require BRCA1 or CtIPImmortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h.Figure 1A Immortalized Brca1F/−MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 and tubulin were used as loading controls. *non‐specific band. B Cells treated as in (A) were fixed 48 h after selection and stained with an anti‐53BP1 antibody (green). Telomeres were visualized with a Cy3‐conjugated (CCCTAA)3‐PNA probe (red). Yellow arrowheads point to 53BP1 foci that co‐localize with telomeres.C Quantification of TIFs in cells treated as in (B). A minimum of 200 nuclei were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05.
Features of ApCatSper 1–4, β, γ, and δ and localization of CatSper in A.Figure 1A Predicted membrane topology and predicted molecular weight of ApCatSper 1-4 cloned from A. punctulata testis, and of ApCatSper β, δ, and γ. A gene encoding ApCatSper δ was identified in the A. punctulata genome; transcripts encoding ApCatSper β and ApCatSper γ were identified in the A. punctulata transcriptome. S1 to S6, transmembrane segments; +, positively charged amino acids in S4; gray cylinder, coiled‐coil domain. B Upper panel, alignment of pore regions of ApCatSper 1-4. Amino acids of the Ca2+ selectivity‐filter motif are highlighted. Lower panel, alignment of S4 voltage‐sensor segments of ApCatSper 1-4 and of D. melanogaster ShakerB Kv channel. Positively charged amino acids are highlighted. Numbers indicate start and end position of amino acids of the selected region. C Western blots of total protein of CHO cells transfected with ApCatSper 2 or 3, non‐transfected control cells (‐), and A. punctulata sperm (Sp). The Western blots were probed with anti‐HA, anti‐ApCatSper 2, or anti‐ApCatSper 3 antibodies. Arrows indicate bands representing ApCatSper 2 and 3. D Immunocytochemistry analysis of sperm stained with anti‐ApCatSper 3, anti‐GC, or anti‐CNGK antibodies; superposition of images obtained by fluorescence microscopy and bright‐field light microscopy; scale bar = 10 μm. The DNA was stained with DAPI (blue). E Western blot analysis of co‐immunoprecipitation (IP) of A. punctulata sperm proteins. The input (I), flow through (FT), washes (W1‐5), and the eluate (E) of the IP using the anti‐ApCatSper 2 antibody were probed with the anti‐ApCatSper 3 antibody (upper panel) and vice versa (lower panel). ApCatSper 3 and ApCatSper 2 were co‐immunoprecipitated with the anti‐ApCatSper 2 (upper panel) and anti‐ApCatSper 3 antibody (lower panel), respectively. F Analysis by mass spectrometry of immunoprecipitated proteins. ApCatSper 1‐4, β, γ, and δ were identified in the immunoprecipitates obtained with both the anti‐ApCatSper 2 and anti‐ApCatSper 3 antibodies. The number of tryptic peptides identified and the respective sequence coverage are given.
Figure 2(A) NMR structure of Ca2+/CaM (grey) bound to unphosphorylated PSD-95. PSD-95 residues D2 (depicted as starting point) to R13 are resolved and form an -helix (cyan) (Zhang et al., 2014). Residues 14-19 of unphosphorylated PSD-95 are unstructured and not shown. Ca2+ ions bound to CaM are depicted by orange spheres. (B) NMR structure of Ca2+/CaM bound to PSD-95 phosphorylated at T19 (PDB accession no. 5J7J). Residues 14-19 of phosphorylated PSD-95 adopt a hook-like structure (highlighted in red) not seen in unphosphorylated PSD-95. This hook-like structure is stabilized by PSD-95 residues E17 and pT19 forming salt bridges with R126 and K115, respectively. Side-chain atoms are depicted by a stick representation for PSD-95 residues Q15, E17 and T19 (red) and CaM residues K115, T117, and R126 (blue). (C) Intermolecular contacts observed for residues in the helical segment of PSD-95_pT19 (side-chain atoms shown as stick representation) and residues in CaM (represented by the colored ovals).
Figure 2a, t-SNE representation of the four subclasses of cancer-associated fibroblasts (CAFs) and perivascular-like cells (PVL), named myofibroblast-like CAFs (myCAFs; 280 cells), inflammatory-like CAFs (iCAFs; 1,129 cells), differentiated-PVL cells (dPVL cells; 122 cells) and immature-PVL cells (imPVL cells; 198 cells). b, Plot showing the proportion of the four stromal subsets across all five patients. c, Log expression of parenchymal gene markers commonly associated with CAFs and perivascular cells. d, Cluster averaged log normalised expression of the top 300 differentially expressed genes between the four stromal subsets with stromal-related genes of interest annotated. Expression values are scaled per cluster. e, Circle histogram plot of the top gene-ontologies enriched in each of the four stromal subsets, with pathways broadly grouped for ECM, development and signalling, muscle contractile-features and angiogenesis and adhesion. Scale bar represents the -log10 q-value for the enrichment of individual GO terms, as determined using ClusterProfiler.
Figure 3Phenotypic analysis. WT, GR-UVR8R338A/uvr8 and GR-UVR8W285F/uvr8 seedlings were grown in LD (16-h light/ 8-h dark) for 5 days, then transplanted to new medium with 0.4 μM NAA and with or without 20 μM DEX and kept in white light plus UV-B (1 W/m2) for 7 days. Images are shown in (A); scale bars = 2 mm. The lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (C) of indicated genotypes were measured. SDs (n > 8) are indicated. UVR8 inhibited auxin responses under UV-B in a tissue-autonomous way. WT and uvr8 seedlings grown in LD for 5 days were used for reciprocal grafting. Seedlings were kept in LD for 7 days after grafting, then transplanted to new medium containing 0.4 μM NAA and kept in UV-B light (1 W/m2) for 10 days. Images are shown in (D); scale bar = 2 mm. The lateral root density (number of lateral roots/length of primary root) (E) and average length of lateral roots (F) of indicated genotypes were measured. SDs (n > 8) are indicated.