EMBO/sd-character-level-ner huggingface dataset:
Figure 2Peak SARS-CoV-2 replication precedes innate immune activation. (A-I) (A,C) Representative images of NF-κB p65 (A) (red) and IRF3 (C) (red) nuclear localisation in mock or SARS-CoV-2 infected (MOI 0.4 TCID50VERO/cell) Calu-3 cells at 24 hpi. SARS-CoV-2 N protein (green. (E and G) Representative images of IL-6 mRNA (E) detected by FISH (red) and N protein (green) , or IFIT1 mRNA (G) (green) with N protein (red), both with nuclei (DAPI, blue) in mock or SARS-CoV-2 infected (MOI 0.4 TCID50VERO/cell) Calu-3 cells at 24 hpi. (B, D, F, H, I) Single cell analysis time course quantifying the Integrated Nuclear Intensity of NF-κB p65 (B), IRF3 (D) , or overall integrated intensity for IL-6 (F) or IFIT1 (H) mRNA over time in N protein positive cells and N protein negative cells (I). n=2. Kruskal-Wallis test with Dunn's multiple comparison. * (p<0.05), **** (p<0.0001). Scale bar represents 50µM. (J,K) Secretion of CXCL10 (J) and IL-6 (K) by infected Calu-3 cells (MOIs 0.08, 0.4 and 2 TCID50VERO/cell), (ELISA). (L) Lactate dehydrogenase (LDH) release into culture supernatants by mock and SARS-CoV-2 infected Calu-3 cells (MOIs 0.08, 0.4 and 2 TCIDVERO50/cell) quantified absorbance (492nm). (M) Quantification of live/dead staining of non-adherent cells recovered from supernatants of mock or SARS-CoV-2 infected Calu-3 cultures at 48 and 72hpi. Data information: (J-M) Means from replicate wells shown +/- SEM, n=2, representative of three independent experiments.
Cysteamine treatment does not rescue CMA impairment in Ctns−/− mice.Figure 11Cystine content in WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers was assessed by mass spectrometry. Results are mean ± SEM (n = 4). ***P < 0.001; **P = 0.0026 (one-way ANOVA, Bonferroni's multiple comparisons test). Western blot analysis of LAMP2A expression in lysosomes isolated from WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers. Lysosomes, isolated as in (B), were incubated with the CMA substrate GAPDH, and CMA activity was evaluated as in Fig8 and as described in Materials and Methods.Quantitative analysis of substrate degradation is expressed as percent of the residual amount of GAPDH in the full reaction relative to its amount in the presence of protease inhibitors. Results are mean ± SEM (n = 4). *P = 0.0126 for WT versus Ctns−/− and *P = 0.0436 for WT versus Ctns−/− + cysteamine; NS, not significant; unpaired t-test.
RAD54 oligomerization and ATPase activity regulate break-induced replication at ALT telomeres A Representative images of combined EdU staining (Click-it) and DNA FISH for telomeres.Figure 3Representative images of combined EdU staining (Click-it) and DNA FISH for telomeres. HuO9 or SaOS2 cells were transfected with 20nM siRAD54#2 (representative images for siRAD54#1 not shown) for 48 hours followed by a 1.5 hr pulse of EdU. Pan-nuclear EdU stain represents S-phase cells. White arrows indicate non-S-phase cells containing EdU colocalizing with telomeres. Scale bars = 10 µm Quantification of data shown in A. HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#1 or 20nM siRAD54#2 for 48 hours followed by a 1.5 hr pulse of EdU. Cells with pan-nuclear EdU signal or greater than 8 EdU foci were excluded from analysis as S-phase cells. Non-S-phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres. Data were normalized to mock condition for each repeat. At least 100 non-S-phase cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Dunnett's multiple comparison test. Representative DNA dot blot from C-circle assay on HuO9 cells, either mock transfected or transfected with 20nM siRAD54#2 for 48 hours. Quantification of C-circle assay in C. Signal was quantified with densitometry, background (- Φ29) was subtracted and signal was normalized to mock. N=5 biological replicates values shown are mean±sd. Conditions were analyzed using unpaired two-tailed student's t-test. Representative images of combined IF and DNA FISH for PML (IF) and telomeres (FISH). HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#2 for 48 hours. White arrows indicate large colocalization events. Blue arrows indicate small colocalization events. Scale bars = 10 µm Quantification HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#1 or 20nM siRAD54#2 for 48 hours. Cells were considered positive if they contained 1 large or 3 small colocalization events. At least 100 cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Dunnett's multiple comparison test. Representative images of combined EdU staining (Click-it) and DNA FISH for telomeric DNA. HuO9 cells were forward transfected with 2µg empty vector (EV), RAD54-WT, RAD54-K189R, or S49E construct. After 24 hours, cells were transfected with 20 nM siRAD54#2 for 48 hours followed by a 1.5 hr pulse of EdU. White arrows indicate non-S-phase cells containing EdU colocalizing with telomeres. Scale bars = 10 µm Quantification of data shown in G. Non-S-phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres. Data were normalized to EV-Mock condition for each repeat. At least 100 non-S-phase cells were counted per condition per repeat, n=4 for EV, WT, S49E, n=3 for K189R. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Sidak's multiple comparison test.
Fig.Figure 5Representative in vitro phase-contrast (A), TH/DAPI or TH (insets) (B), and TH/SYNAPSIN-stained (C) images in the cultures terminally differentiated from mutant (c13, c19) and corrected hiPSCs (c22, c59). DA neuron differentiation of DA neurons were assessed by DA neuronal yields DA neuron morphological of DA neurons were assessed by TH+ fiber lengths DA neuron , synaptical of DA neurons were assessed by synaptic puncta densities DA neuron differentiation functional maturation of DA neurons were assessed by presynaptic DA neuron releases DA levels were measured in the media conditioned in the differentiated cultures for 24 h (D15) and evoked by KCl-mediated depolarization for 30 min. DNA breaks estimated by γH2AX staining. Cells with >4 γH2AX foci in the nucleus were counted. SA-β-gal staining was performed for cellular senescence analysis. Mitochondrial ROS (MitoSox) were analyzed after terminal differentiation. mitochondrial membrane potential (MitoID) were analyzed after terminal differentiation. Accumulation of α-synuclein oligomers analyzed by western blotting. The mutant (c13) and corrected (c22) neural stem cells (NSCs) were transduced with α-synuclein-expressing lentiviruses. At 6 days after differentiation, cells were extracted and fractionated to Triton X-100 (1%)-soluble and insoluble fractions. Shown is the representative WB image of three independent experiments. α-Synuclein oligomers are indicated with a bracket, while monomer bands are marked with an arrowhead. ClueGO/cytoscape for GO biological process upregulated in NSCs differentiated from the corrected iPSCs, c22 (vs the mutant pt-hiPSCs, c19). Term enrichment significance is represented by node size (Kappa score > 0.4). Most significant GO-terms are numbered and listed below.
Figure 5A-C The cation concentrations used are indicated for each experiment; AU, arbitrary units.
a,b) Electron micrographs of motor neuron perinuclear regions from age-matched non-transgenic (Nt), YAC AR20 and YAC AR100 transgenic mice before disease onset (a, 6 months of age) and after prominent neuromuscular and molecular pathology is apparent (b, 14 months of age).figf2(a,b) Electron micrographs of motor neuron perinuclear regions from age-matched non-transgenic (Nt), YAC AR20 and YAC AR100 transgenic mice before disease onset (a, 6 months of age) and after prominent neuromuscular and molecular pathology is apparent (b, 14 months of age). (a) At 6 months of age, occasional autophagosomes (yellow arrowheads) are noted in Nt, YAC AR20 and YAC AR100 at roughly equivalent frequency. Autolysosomes (red arrowheads) are much more common and are present in higher numbers in YAC AR20 and YAC AR100 motor neurons. (b) At 14 months of age, when YAC AR100 mice display signs of motor neuronopathy and molecular pathology, we observed many YAC AR100 motor neuron micrographs with frequent autophagosomes (yellow arrowheads). Despite this increase in autophagosomes, autolysosomes were fewer in number at this age in YAC AR100 motor neurons, such that autophagosome numbers approached autolysosome numbers in YAC AR100 motor neurons. This was never the case for Nt motor neurons or YAC AR20 motor neurons. Main panels are at original magnification 2,200× and insets are at original magnification 3,700×.
Figure 2Content of soluble sugars in guard cell-enriched epidermal peels of WT and stp1stp4 plants at the end of the night (EoN) and after 40 min of illumination with white light at 150 µmol m-2 s-1 following the EoN. Data for two independent experiments are shown; means ± SEM; n ≥ 7 per genotype and time point. Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1 and suc3 plants. Scale bar, 10 µm. Starch dynamics in guard cells of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1 and suc3 plants at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for three independent experiments are shown; means ± SEM; n = 120 individual guard cells per genotype and time point.
Figure 1A) Chemical structures of LUF7243, LUF7244, LUF7344, LUF7346 and LUF7389. B) Percentage specific binding of [3H]dofetilide to the hERG channel after 6 minutes of dissociation induced by 10 µM dofetilide in the absence (control) or presence of 10 and 50 µM of LUF compounds. The specific binding of [3H]dofetilide in the absence of test compounds was set as Bcontrol, while the specific binding in their presence was set as B. * = p < 0.05 vs control; N: 3-4. C) Representative traces of hERG activation (left), inactivation (middle), and deactivation (right) measured in HEK293 hERG cells under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). Insets: voltage clamp protocols. D) Steady-state activation (empty symbols) and inactivation (filled symbols) curves for IKr under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). The corresponding Boltzmann's fittings are superimposed to data points.* = p < 0.05 vs baseline. N: 11-14. E) Plot of the time constants (τ) of deactivation derived from biexponential fittings under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). * = p < 0.05 vs respective baseline. N: 8.
The pneumococcal CbpC protein can activate autophagy, but can also facilitate intracellular pneumococcal survival.Figure 1A Pneumococcal CBPs used in (B). B Confocal images of HeLa cells transiently expressing GFP-CBPs or GFP. The dotted lines show each cell shape. Scale bars, 10 µm. C Confocal images of HeLa cells transiently expressing GFP-CbpCT4 and mCherry-LC3 (upper). The fluorescence intensities of GFP-CbpCT4 (green) and mCherry-LC3 (red) along the arrow are shown in the graph at the bottom. D Lysates from 293T cells transiently expressing GFP-Cbps or GFP were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin. E Lysates from 293T cells transiently expressing GFP-CbpCT4, GFP-LytR, or GFP in the presence or absence of chloroquine were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin. F MEFs were infected with S. pneumoniae R6 WT or ΔcbpF for the indicated periods, and the intracellular survival of bacteria expressed as the number of CFUs. G p62-KO MEF cells infected with S. pneumoniae R6 ΔcbpF/pCbpFR6-FLAG for 2 h were fixed and stained with DAPI and an anti-FLAG antibody, and representative epifluorescence images are shown. Scale bars, 10 µm.
Figure 5(A-C) Thymi from E18.5 embryos are shown: (A) intact with genotypes indicated, or (B, C) after sectioning, immunofluorescent staining for DLL4 (red), and counterstaining of DNA (blue). Sectioned samples are wild-type (B) or Aff4ko/ko; Foxn1+/- (C). Scale bars: A, 1 mm; B-C, 50 µm.
Conditional deletion of Wnt4 in the mammary epithelium and its consequences A–D Representative epifluorescence stereo micrographs of inguinal mammary glands from a 10‐day‐old mT/mG (A, C) and MMTV::Cre; mT/mG (B, D) female (n = 7).Figure 3A-D Representative epifluorescence stereo microscopy of inguinal mammary glands from a 10‐day‐old mT/mG (A, C) and MMTV::Cre; mT/mG (B, D) female (n = 7). Scale bar: 1 mm. E Higher magnification of inguinal mammary gland from a 10‐day‐old MMTV::Cre; mT/mG female. Scale bar: 0.2 mm.F Immunofluorescence microscopy for EGFP (green) on a mammary gland section from a 10‐day‐old MMTV::Cre; mT/mG female counterstained with DAPI (blue) (n = 7). Scale bar: 30 μm. G Epifluorescence stereo microscopy of mammary glands from 10‐day‐old littermates either MMTV::Cre; mT/mG Wnt4fl/+ or MMTV::Cre; mT/mG; Wnt4fl/fl. Scale bar: 1 mm. Arrowhead marks the main duct originating from the nipple.H Bar plot showing ratio of branching points in prepubertal Cre+;Wnt4fl/fl (n = 12) relative to Cre+;Wnt4fl/+ littermates (n = 10). Two‐tailed, paired Student's t‐test was used to calculate statistical significance. I Fluorescence stereo microscopy of mammary glands from 35‐day‐old littermates either MMTV::Cre; mT/m; Wnt4fl/+ or MMTV::Cre; mT/m;Wnt4fl/fl. Note ductal elongation is delayed in Wnt4‐deficient mammary epithelium compared to Wnt4fl/+ control. LN: lymph node. Scale bar: 4 mm.J, K Bar plots showing number of terminal end buds (TEBs) (J) and area of mammary fat pad filled by the ductal system (K). Ctrl (Wnt4fl/wt or Wnt4fl/fl; Cre−): n = 6, fl/+; Cre+: n = 4, and fl/fl; Cre+: n = 7). Data are presented as the mean ± SD. Two‐tailed Student's t‐test was used to calculate statistical significance. L, M Bar plots showing BrdU incorporation index in inguinal mammary glands of 4‐week‐old Wnt4fl/+; Cre+ (n = 3) or Wnt4fl/fl; Cre+ (n = 4) in the ducts (L) and in the TEBs (M). Data are presented as the mean ± SD. Two‐tailed Student's t‐test was used to calculate statistical significance.
VAMP3 Regulates mATG9 and ATG16L1 Trafficking.Figure 6(A-C) HeLa cells transfected with control, VAMP3 siRNA for 4 days were transfected during the last 20 hr with ATG9L1-pEGFP and labeled with anti-EEA1 (for early endosomes) or anti-RAB11 (for recycling endosomes). The histogram in (C) shows the quantification of the colocalization of mATG9 in EEA1 or RAB11 (Manders' coefficient). Error bars, SEM. ∗∗∗p < 0.001. (D-F) HeLa cells were treated as in (A)-(C) and transfected during the last 20 hr with pEGFP-ATG16L1 and labeled with anti-EEA1 (early endosomes) or anti-RAB11 (recycling endosomes). Histogram in (D) shows quantification of the colocalization of ATG16L1 in EEA1 or RAB11 (Manders' coefficient). Error bars, SEM. ∗∗∗p < 0.001; NS, not significant.
Inhibition of the Small GTPase rab-10 Increases Autophagy.Figure 5(A) Average number of LGG-1::GFP-containing puncta in N2 wild-type animals (WT) fed either control bacteria or bacteria expressing daf-2 (as a control) or rab-10 dsRNA for two generations. p < 0.0001 for either daf-2 or rab-10 RNAi treatment compared to control RNAi treatment, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Please see Table S1 for quantification of all data.(B) Average number of LGG-1::GFP-containing puncta in rab-10(ok1494) mutants compared to N2 wild-type animals (WT), p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. Please see Figure 1 for details. Please see Table S1 for quantification of all data. (C) Survival curves of rab-10(ok1494) animals fed either control bacteria or bacteria expressing bec-1 or vps-34 dsRNA during adulthood at 20 °C. Mean lifespan was 27.9 d for control, 19.9 d for bec-1 RNAi, and 22.1 d for vps-34 RNAi, all pair-wise comparisons to control, p < 0.0001, Log-rank (Mantel-Cox) test. This experiment was performed two times. Please see Table 3 for additional data.(D) Survival curves of N2 wild-type animals (WT) fed either control bacteria or bacteria expressing bec-1 or vps-34 dsRNA during adulthood at 20 °C. These assays were performed at the same time as the rab-10 lifespan analysis shown in Figure 5C. Mean lifespan was 22.9 d for control, 23.6 d for bec-1 RNAi, and 24.5 d for vps-34 RNAi, pair-wise comparison to control, p = 0.057 and p = 0.16, respectively, Log-rank (Mantel-Cox) test. Please see Table 1 for additional data.
Biochemical properties of PrPSc assessed by guanidine denaturation and CDI.Figure 7A Spot counts in the scrapie cell assay using WT, TgPrP(S1)-17 and TgPrP(S3.F88W)-14 brain homogenates (0.1% or 0.01%) from mice infected with RML are shown in the upper panel (WT vs S1 P = 2.09E-05 and vs S3 P = 4.62E-08) with numbers corrected for PrPC expression levels as per Table1 presented in the lower panel (WT vs S1 P = 1.04E-13 and vs S3 P = 3.39E-20; two-tailed t-test, n = 3). B A comparison of incubation time vs PrPC:PrPSc ratio for mice expressing different levels of WT PrP (WT, TgPrP(WT), tga20), S1 PrP (TgPrP(S1)-17, TgPrP(S1)-19, TgPrP(S1)-39) and S3 PrP (TgPrP(S3.F88W)-14, TgPrP(S3.F88W)-35). C Formation and biological activities of PrP species. Synthetic and proteolytic pathways are shown from left to right, while pathways for pathologic signalling from PrPSc through PrPC and PrPC-derived fragments are shown from right to left (dashed arrows). Alternative pathways to remove N-terminal sequences (yellow) from the protease-resistant C-terminal Core (red) in infected cells are shown, while N2 is removed from a protease-sensitive C-terminal core (blue) in healthy cells. Grey arrow indicates that formation of PrPSc C2 from protease-sensitive C2 is not particularly efficient as mice with high levels of C2 are not more efficient in synthesizing PrPSc. Heavy dashed arrow through protease-sensitive C2 indicates that this species may be adept at pathogenic signalling as mice with lower PrPSc burden than WT mice succumb to disease earlier. For simplicity, physiological signalling pathways through PrPC species are not shown.
Figure 3Schematic representation of a SILAC-based mass-spectrometry approach (upper graph). The p97 interactome showing ATX3 as the only DUB in nucleus with increased interaction with p97 after IR (10 Gy; lower graph). Schematic representation of ATX3 domain structure. C14; cysteine catalytic centre at amino-acid 14, UBS1 or 2; ubiquitin binding sites 1 or 2, UIM; ubiquitin interacting motif, VBM; p97 binding motif, poly Q; polyglutamine region. Western blot analysis of Strep-p97 Co-IP in nuclear fraction of HEK293 cells showing interaction of p97 with ATX3 under physiological conditions and after IR (10 Gy). Western blot analysis of Flag-ATX3 Co-IP in HEK293 cells showing interaction of ATX3 with p97, RNF8 and PSα6 under physiological conditions and after IR (10 Gy). Western blot analysis of Flag-ATX3 WT or Flag-ATX3 VBM* Co-IP in HEK293 cells showing interaction of ATX3 with p97, RNF8 and p97 core co-factors Npl4-Ufd1. (* represents unspecific bands). Western blot analysis of Strep-p97EQ Co-IP in HEK293 cells, in presence or absence (CRISPR knockout; ∆ATX3) of ATX3, showing increased interaction of RNF8 with p97 in absence of ATX3, under physiological conditions and after IR (10 Gy).   Graph represents quantifications of (F) (*P<0.05, **P<0.01; unpaired t-test, n=3, mean +SEM).   Model showing the interaction of p97 and ATX3 with ubiquitinated RNF8.
a) Aversive associative memory performance 3 min after training (STM) was markedly reduced in 3-d-old Atg7−/− flies (Atg7d14/Atg7d77) compared with Atg7−/+ flies (Atg7d14/CG5335d30) and wild-type flies (Atg7+/+) (n = 8–11 independent experiments, F = 25.figf5(a) Aversive associative memory performance 3 min after training (STM) was markedly reduced in 3-d-old Atg7−/− flies (Atg7d14/Atg7d77) compared with Atg7−/+ flies (Atg7d14/CG5335d30) and wild-type flies (Atg7+/+) (n = 8-11 independent experiments, F = 25.82, one-way ANOVA with Bonferroni correction). The CG5335d30 line harbors an Atg7+ chromosome related to Atg7d14 and Atg7d77 that serves as a genetic background control. (b) Olfactory learning was disrupted in 3-d-old female flies homozygous for Atg8a hypomorph (Atg8a−/− or Atg8aEP362/Atg8aEP362) (n = 8-9 independent experiments, F = 22.8, one-way ANOVA with Bonferroni correction). (c) STM is severely impaired in 20-d-old Atg7−/− (Atg7d14/Atg7d77) flies and 20-d-old Atg7−/+ heterozygous (Atg7d14/CG5335d30) when compared to age-matched wild-type flies (n = 8-9 independent experiments, F = 18.02, one-way ANOVA with Bonferroni correction). (d) Similarly, STM was markedly decreased in 20-d-old Atg8a−/− (Atg8aEP362/Atg8aEP362) mutant female flies compared with control flies (Atg8a+/+) without any effect by spermidine feeding (n = 8-9 independent experiments, F = 68.43, one-way ANOVA with Bonferroni correction). *P 0.05, **P 0.01, ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m. The aversive olfactory avoidance and shock reactivity of different mutant are shown in Supplementary Table 5.