EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 8A) Representative images from live-cell imaging of siRNA transfected B1dd/B2ko cells (FusionRed-H2B in red, SiR-Tubulin in white, scale bar = 10µm). Bar-plot panels on the right show single-cell analysis of 10 cells manually scored for length mitosis pre-NEBD and post-NEBD. Entry into prophase was scored by cell rounding and NEBD was identified by influx of Tubulin in the nucleus. (B) Widefield Imaging. DIC (Grey), SiR-DNA (b/w), of ENSA/ARPP19 siRNA transfected DIA treated B1dd/B2ko cells. Time is indicated in hh:min, scale bar = 10µm. (C) Panels of representative immunofluorescence images of siRNA transfected and P/A treated cells treated as indicated, scale bar = 10µm. (D) Quantification of mitotic index of siRNA transfected P/A synchronised cells (E) Quantification of mitotic B1dd/B2ko cells following siRNA transfection to deplete Greatwall (siGwl) or ENSA/ARPP19 (siE/A) and/or DIA treatment. Frequencies of mitotic cells with intact (blue) and disassembled (red) Lamin A/C staining are plotted (mean value of three repeats, n>50 per repeat, error bars indicate standard deviation). quantification of control and DIA treated mitotic B1dd/B2ko cells following siRNA transfection to deplete Greatwall and B55α/δ. Frequencies of mitotic cells with intact (blue) and disassembled (red) Lamin A/C staining are plotted (mean value of three repeats, n>50 per repeat, error bars indicate standard deviation). (G) Intensity of pSP-antibody staining in siRNA transfected and DIA treated B1dd/B2ko cells. The swarm-blots classify data from mitotic cells with intact (blue) or disassembled (red) Lamin A/C. (H) As in (G) Intensity of anti-Lamin A/C pS22 antibody staining in siRNA transfected and DIA treated B1dd/B2ko cells. (I) Same as in (G) and (H), data for centrosome distance in mitotic cells. Data for G-I are from three repeats, n>50 per repeat, the bars indicate median, first and third quartile as well as minimum and maximum values, data are from three repeats, n=50 cells per repeat).
A.Figure 4A. Protein extracts corresponding to tachyzoites incubated in the same starvation conditions as in Figure 2B were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect TgAtg8 and the lipidated TgAtg8-PE form. GFP-TgAtg8 parasites extracts were analysed with anti GFP antibody (left), while parental cell line was analysed with anti-TgAtg8 antibody (right). Anti-ROP5 was used as a loading control. B. Cell lysates from GFP-TgAtg8 parasites were subjected to a centrifugation at 100,000 g to separate the soluble fraction (high speed supernatant, HSS) from the membrane fraction (high speed pellet, HSP). The faster migrating form of GFP-TgAtg8 is exclusively present in the membrane fraction as revealed by Western blot analysis after urea SDS-PAGE using anti-GFP C. The HSP fraction described in B. was extracted with various agents and new HSS and HSP fractions were separated and analysed by Western blot after urea SDS-PAGE. Only a treatment with a detergent (DOC) resulted in GFP-TgAtg8 solubilisation from the HSP fraction. D. GFP-TgAtg8-expressing parasites were grown in host cells in the presence of 3H-ethanolamine and then starved in HBSS for 8 hours, still in the presence of the radioactive PE precursor. Tachyzoites were then lysed and GFP-TgAtg8 was immunoprecipitated using anti-TgAtg8 antibody and analysed by urea SDS-PAGE. The immunoprecipitated forms of GFP-TgAtg8 were detected by Western blot using anti-GFP antibody and radioactive ethanolamine was found to be incorporated into immunoprecipitated GFP-TgAtg8, as detected by fluorography (3H-etn).
Figure 4a. Sorted CD4+CD25hiCD127- were pre-incubated with increasing concentrations of AS1842856, washed and co-cultured with CFSE-labeled responder T cells. Representative histograms of proliferation of Tresp alone (left) or in co-culture with Tregs treated with either vehicle or different concentrations of AS1842856 measured at day 4 at a 1:2 Treg:Tresp ratio. b. Statistical analysis of the suppression of proliferation at different Treg:Tresp ratios (n=6) on Tregs pretreated with vehicle (white dots) or 10 nM (black dots, left diagram) or 50 nM AS1842856 (black dots, right diagram). c. Representative dot plots of the frequency of IFN+Foxp3+ Tregs in the co-cultures in a at a 1:2 Treg:Tresp ratio after adding GolgiStop for the last 6 hours. d. Statistical analysis of the frequency of IFN+Foxp3+ Tregs in the co-cultures in a of n=6 experiments performed. e. Correlation between frequency of IFN+Foxp3+ Tregs in co-cultures and percentage of suppression in Tregs pre-incubated with different doses of AS1842856 at a 1:4 Treg:Tresp ratio. f. IFNG and TBX21 gene expression on Tregs transduced with a Foxo1 or Foxo3 shRNA as compared to the NT-shRNA control, measured at day 6 after 4 hours of stimulation with PMA and ionomycin. g. Percentage of suppression of CFSE-labeled Tresp proliferation co-cultured with Tregs transduced with a non-target (white dots), Foxo1 (left diagram, black dots) or Foxo3 shRNA (right diagram, black dots) at different Treg:Tresp ratios (n=4). *p < 0.05, **p < 0.005, ***p < 0.0005.
| Monovalent antigen controls IgD BCR activation A | Schematic overview of reconstitution of BCR-deficient Ramos (HL-KO) cells with murine HEL-specific BCR of IgM (HH10-mu IgM) and IgD (HH10-mu IgD) isotype, respectively.Figure 5A | Schematic overview of reconstitution of BCR-deficient Ramos (HL-KO) cells with murine HEL-specific BCR of IgM (HH10-mu IgM) and IgD (HH10-mu IgD) isotype, respectively. B | Representative flow cytometric analysis of Ramos cells reconstituted with HH10-mu IgM, HH10-mu IgD. Cells were stained with α-murine μHC-, δHC- and κLC-antibodies, respectively. EV-transduced HL-KO cells, expressing GFP only, were used as negative control (gray). C | Representative flow cytometric analysis of HEL-binding in reconstituted Ramos cells after staining with fluorescently-labeled HEL. EV-transduced HL-KO cells, expressing GFP only, were used as negative control (gray). D | Representative intracellular Ca2+ influx in HH10-mu IgM- and HH10-mu IgD-expressing cells upon stimulation with multivalent HEL (complex cHEL), monovalent (soluble sHEL) (both at a concentration of 1 μg/ml), or 10 μg/ml α-mouse κLC antibody, respectively. E | Representative intracellular Ca2+ influx of HH10-mu IgM- (top) and HH10-mu IgD- (bottom) expressing cells upon stimulation with indicated ratios of 1 μg/ml cHEL and sHEL, and a 1 : 25 mixture of cHEL with bovine serum albumin (BSA), where 1 = 1 μg/ml. F | Schematic overview of reconstitution of BCR-deficient Ramos (HL-KO) cells with HH10-specific human IgM (HH10-hu IgM) and IgD (HH10-hu IgD) BCR isotype. G - J | using HH10-hu IgM- and HH10-hu IgD-expressing cells instead of HH10-mu IgM- and HH10-mu IgD-expressing cells, respectively. Data shown in G to J are representative of minimum three independent experiments.
Micrograph of nerve cells of Porcellio d.Figure 4Micrograph of nerve cells of Porcellio d. dilatatus injected by wVulC.These cells are localised in the CNS ganglion. (A) At 30 days post-injection, some autolysosomes and bacteria are visible: autophagic process is already quite high compared to control [N: Nucleus, V: Vacuole, Ap: Autophagosomes (Autophagic hallmark), Al: Autolysosomes, Wo: Wolbachia, *: a Wolbachia which seems to be surrounded by an autolysosome, Arrow head: magnified area]. (B) At 60 days post-injection, the nerve cells are severely damaged by the accumulation of autophagic vesicles (N: Nucleus, V: Vacuole, Ap: Autophagosomes (Autophagic hallmark), Al: Autolysosomes, Arrow head: magnified area and probably a Wolbachia in autophagic degradation).
Figure 1A Domain composition of full-length titin (top) and detail of the A168-M1 kinase region in the sarcomeric M-line (bottom). Domain color-coding in the latter is maintained throughout this manuscript. Various components of full-length titin are annotated to serve as markers. The MuRF1 binding site in the multi-domain kinase region is indicated. B Crystal structure of A170-M1. C Close-up view of the packing of the NL sequence against TK (left). Residues in the NYD motif are displayed. The conserved aspartate residue affected by the D24728V exchange in SNP rs200675195 is marked in magenta. Directed interactions established by NYD residues with other parts of the A170-M1 protein are shown in the inset (right) as dashed lines. These interactions are the primary anchor of the NL element onto TK.
Integrated Systems Analysis Identifies KLHL9, NEDD8, and KLHL13 as CLEC12A-Interacting Antibacterial Autophagy Molecules.Figure 6(A) Gene expression analysis of WT and Clec12a−/− BMDMs infected with Listeria for the indicated times. Heatmap shows log2 (fold change) relative to uninfected WT BMDMs at indicated time points. Shown are the top ten most-significant upregulated and downregulated genes in Clec12a−/− BMDMs compared to WT BMDMs. (B) Cluster analysis of 674 differentially expressed genes in Clec12a−/− BMDMs versus WT BMDMs. (C) Schematic of integrated proteomic and transcriptomic approach to identify functionally relevant CLEC12A-interacting proteins. (D) Interactome analysis shows network of proteins identified from quantitative iTRAQ proteomics. Red box highlights the KLHL9-KLHL13-CUL3-NEDD8 complex interaction within the CLEC12A interactome. (E) HeLa GFP-LC3 cells were transfected with indicated siRNAs for 48 hr and then infected with DsRed-labeled Salmonella for 1 hr. The fraction of GFP-positive intracellular Salmonella is shown. Data are mean ± SD and are representative of three independent experiments. (F) Gentamicin protection assay in HeLa cells showing increased intracellular bacterial replication after 48 hr of knockdown of ATG16L1, KLHL9, NEDD8, or KLHL13. Data shown as mean ± SD; n = 4. Data are representative of three independent experiments.
Figure 1A) A 137 kb region encompassing exons 18-30 was targeted with 4 sgRNAs in introns 17 and 30 to generate a genomic duplication. Cas9 and the sgRNAs are represented as scissors. B) Schematics of the 12049 bp inversion present in the Dup18-30i mice and of the strategy utilized to correct the inversion. Two sgRNAs targeting the inversion were utilized to rescue the complex rearrangement. Cas9 and the sgRNAs are represented as scissors. C) PCR amplification and Sanger sequencing of the duplication junction in the Dup18-30 founder mouse confirming the joining of intron 30 and 17, along with a 96 bp intronic deletion. D) The heart, tibialis anterior (TA) and triceps muscle were isolated and analyzed to identify the presence of the duplication from exons 18 to 30 via RT-PCR using primers represented by the arrows. Sanger sequencing confirmed correct splicing of exons 30 and 18 at the duplication junction. E) Protein lysates isolated from the TA, triceps, diaphragm and heart muscles of WT and Dup18-30 mice were probed for dystrophin expression by western blot. Calnexin serves as a loading control.
PINK1 recruits cytoplasmic Parkin to damaged mitochondria.Figure 4.(A) PINK1 knockout (PINK1−/−) or control (PINK1+/+) MEFs were transfected with HA-Parkin, treated with CCCP, and subjected to immunocytochemistry with the indicated antibodies. Higher magnification views of the boxed areas are shown in the insets. (B) The number of MEFs with Parkin localized to the mitochondria was counted as in Fig. 3 A. (C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 4 h and subjected to immunoblotting (C) (C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 24 h, followed by immunocytochemistry (D). IB, immunoblot; Ub, ubiquitylation. (E) The number of MEFs without anti-Tom20 antibody-detectable mitochondria was counted as in Fig. 3 A. In the example figure (left), blue arrowheads indicate cells without anti-Tom20 antibody-detectable mitochondria, and red arrowheads indicate cells harboring anti-Tom20 antibody-detectable mitochondria.
Figure 4A Immunofluorescence of unpermeabilized stably transfected MDCK cells shows that, unlike wt full-length UMOD, mutants ZP-N (R415A) or ZP-C (ΔFA) do not assemble into filaments. Scale bar: 50 µm. B-G Immunofluorescence of unpermeabilized cells co-expressing FLAG-tagged wt UMOD (green) and the indicated HA-tagged isoforms (red). UMOD R415A does not incorporate into polymers that only contain wt protein (C). UMOD ΔFA has a dominant negative effect on wt protein polymerization (D) that is rescued in a double mutant carrying both ZP-N and ZP-C mutations (F). Similarly, the dominant negative effect of a CCS mutation (4A) that prevents EHP dissociation (E) is suppressed by the introducing the R415A mutation in the 4A isoform (G). Scale bar: 50 µm. H Immunofluorescence of permeabilized cells expressing soluble isoforms of UMOD truncated before the EHP (UMOD-CCS) shows that wt forms intracellular polymers whereas polymerization interface mutants do not. The intracellular polymers are localized in the endoplasmic reticulum (ER), as shown by co-staining with the KDEL sequence used as ER marker. Scale bar: 10 µm.
FIP200 is required for autophagy.Figure 5.(A) FIP200+/+, FIP200−/−, Atg5+/+, and Atg5−/− MEFs were cultured in complete or starvation medium for 60 min. In the recovery experiments, starved MEFs were cultured in fresh complete medium for an additional 60 min (replenishment). The cell lysates were subjected to immunoblot analysis with the indicated antibodies. (B) Wild-type and FIP200−/− MEFs were cultured in the complete or starvation medium for indicated time with or without 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody. (C) Wild-type and FIP200−/− MEFs were transfected with retroviral vectors encoding GFP-Atg5 or GFP-LC3. Resulting cells were cultured in the starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm. (D-G) Wild-type (D and E) and FIP200−/− (F and G) MEFs were cultured in complete (D and F) or starvation (E and G) medium for 120 min and then fixed and subjected to EM analysis. Autophagosome-like structures (open arrowheads), and autolysosomes (closed arrowheads) are indicated. Bar, 1 μm. (H) The ratio of total area of autophagosomes (AP) and autolysosomes (AL) to total cytoplasmic area in D-G was determined by morphometric analysis.
Figure 7(A) Phenotype of plants complemented with WT DDL or DDL12E1. Four-week-old plants of two independent transgenic lines of pDDL::DDL-FLAG (W7 and W18) or pDDL::DDL12E1-FLAG(E11 and E15) were photographed. Bar=1cm. (A) Phenotype of plants complemented with WT DDL or DDL12E1. Four-week-old plants of two independent transgenic lines of pDDL::DDL-FLAG (W7 and W18) or pDDL::DDL12E1-FLAG(E11 and E15). The expression of transgene was determined by an α-FLAGimmunoblot (bottom). (B) Susceptibility of complementation lines to Pst DC3000 infections. Four-week-old transgenic plants were inoculated with Pst DC3000 (OD600=5x10-4) and the bacterial number was determined at 0 and 3 dpi. (C) PR1 and PR5 induction in transgenic lines. Leaves of four-week-old plants were inoculated with Pst DC3000 (OD600=0.01), and collected at 0, and 24 hpi for qRT-PCR analysis. The data in B and C are shown as mean ± SD (n=3) from three independent repeats with one-way ANOVA analysis and Tukey test (p<0.05). Different letters indicate significant differences. (D) flg22-induced callose deposition in transgenic lines. Leaves of four-week-old plants were inoculated with 500 nM flg22, and callose deposits were stained with aniline blue at 20 hpi. Number of callose deposits was quantified with Image J (mean ± SE, n=10). All experiments were repeated three times with similar results.
Disomes stalled on poly(A) tracts form a novel POST-POST assembly.Figure 6(A, B) Composite cryo-EM density map (A) of the POST-POST disome stalled on the poly(A) mRNA reporter filtered according to local resolution and used to build the molecular model (B). (C) Cut top views of both the first (stalling) and the second (colliding) ribosomes forming the disome. Observed ribosomal and tRNA translocation states are indicated. (D) Comparison of ribosomal assemblies between the previously described CGA-CCG stalled yeast disome in pink (EMD-4427, Ikeuchi, Tesina et al., 2019a) and the novel POST-POST assembly observed in poly(A) stalling. The EMD-4427 density map was fitted into the density of the first stalling ribosome on the poly(A) reporter. The indicated rotation was calculated using the 60S subunit, as the compared colliding ribosomes are not in the same translocation state (PRE vs. POST).
CDC7 inhibition suppresses Histone H2AX phosphorylation independently from origin firing A.Figure 2A. Outline of experimental procedure. U2OS cells were either untreated, treated with 10 μM XL413 or with 4 mM HU in the presence or absence of XL413 for 24 hours. Cells were EdU labelled for 30 minutes before harvesting. Cells that had been treated with both XL413 and HU for 24 hours, were washed with equilibrated media and retreated for a further 2 hours. All onward treatments included 4 mM HU, to prevent further DNA synthesis either in the absence or presence of CDC7 (XL413), MRE11 (Mirin) or ATR (AZD6738) inhibitors. B. CSK soluble and chromatin-enriched protein fractions were analysed by western blotting with the indicated antibodies. Representative of two independent experiments. C-D. Flow cytometry analysis to assess levels of pS139 Histone H2AX (c). Histograms show the mono-parametric analysis of cell count against pS139 Histone H2AX intensity. Histograms are overlaid to appreciate changes in pS139 Histone H2AX intensity upon treatment (Red lines) relative to appropriate experimental baseline controls (Grey lines). Data are representative of two independent experiments. E. Graphical concept: cells treated with HU and XL413 for 24 hours would have stalled forks, which exist in a stable state due to inhibition of CDC7 and with an active ATR-dependent origin firing checkpoint. Upon washing and retreating of cells in the presence of HU the late origin checkpoint is maintained, in an ATR-dependent manner, and accumulation of DNA damage (pS139 H2AX) can be monitored independently from origin firing in the absence or presence of CDC7 (XL413) or MRE11 (Mirin) inhibitors. AZD6738 was used as a control to investigate molecular events occurring upon loss of checkpoint caused by both fork collapse and loss of the inhibition of origin firing. The increase in chromatin binding of CDC45 was used as a surrogate marker for origin activation, pS139 Histone H2AX was used to monitor fork stability, pS345 CHK1 was used to monitor the ATR-checkpoint signalling and pS40/41 MCM2 was used to monitor CDC7 kinase activity.
Figure 6A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. After calcein injection, the prepared femoral sections of the OVX mice were examined under fluorescence microscope to visualize integrated calcein (A, green, arrows). Nuclei were counterstained with DAPI (A, blue). Osteoblast numbers (B) and calcein double labelled surfaces (C) were measured in the femoral sections. A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. The mouse femurs were analyzed using micro-CT and BMD (D) and BV/TV (E) were calculated from the micro-CT data. A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. The three-dimensional images of femoral trabecular bone were reconstructed (F), and trabecular number (G) and trabecular separation (H) were calculated.
Figure 6A. Experimental setup of inducing aggregation in Aβ biosensor cell line. B. Representative images of Aβ biosensor seeding with 3 different seed concentration (10, 50, 100nM) Scale bars: 100μm C. Treating Aβ biosensor with different concentration of rAβ1-42 seeds induces the aggregation of mCherry-Aβ1-42 in a dose dependent matter (n=3 independent experiments, graph: mean and 95%CI). FRAP of Aβ spots shows limited recovery confirming that are aggregates (in detail at Appendix figure S10b,c). Scale bar: 2.5μm D. Representative images of 3 proteins that can induce aggregation of Aβ1-42 in biosensor cell. Increase aggregation is observed in cells expressing the construct but not GFP alone (left panels). Scale bar: 100μm. FRAP of the resulting aggregates shows no recovery (in detail at Appendix Figure S11). Scale bar: 2.5μm Removal of the homologue regions resulting in reduced aggregation (right panels). (n=3 independent experiments). E. Quantification of number of spots per cell in cells expressing/not expressing the construct (identified by GFP, transfection reporter) (n=3 independent experiments, statistics: ordinary one-way Anova with Dunnett T3 multiple comparison correction, unpaired t-test for transfected/non-transfected cells). Bar plot: mean with 95%CI. * P ≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 F-G. Quantification of Spots per cell for construct6 and construct6 control (6Ctrl, removal of homologue region) (F) and construct10 and construct10 control (10ctrl, removal of homologue region) (G). (n=4 independent experiments, statistics: ordinary one-way Anova). Bar plot: mean with 95%CI. **** P≤0.0001 H-I) Confocal images of colocalization of protein of interest with Aβ aggregates. (Contrast of images were enhanced to 0.1% saturated pixels using Fiji). Scale bars: 22μm
a, Intracellular staining for LC3 and ATG16L1 following glucose starvation in the presence of bafilomycin A1.f3a, Intracellular staining for LC3 and ATG16L1 following glucose starvation in the presence of bafilomycin A1. White mask in LC3 channel illustrates punctate LC3 staining, representing LC3-II. Histogram depicts mean fluorescence intensity of 10,000 cells per sample. b, Quantification of mean ATG16L1 fluorescence intensity in a, n = 4 mice. Glu., glucose. c, Immunoblot of phosphorylated (p-) AMPK&amp;amp;agr; (Thr 172) and lipidated LC3 (LC3-II). d, Quantification of mean LC3-II area in murine macrophages, n = 4 mice. e, Caspase 3 activity in murine macrophages following glucose starvation, n = 3 mice. RLU, relative light units. f, Quantification of mean LC3-II area in wild-type (Casp3+/+) or Casp3-knockout (Casp3−/−) macrophages, n = 3 mice. Data in a-f represent 2 independent experiments.
Special stains of the lungs in the WT-HB-01 and ACE2-HB-01 mice at 3dpi and 5 dpi.Supplementary Figure 1.
MTM‐3 acts downstream of EPG‐8, ATG‐9, ATG‐18, and ATG‐2 in the aggrephagy pathway A–K Confocal fluorescent images of 200‐cell‐stage embryos from the indicated strains stained by both anti‐SEPA‐1 and anti‐LGG‐1 antibodies.Figure 3A-K Confocal fluorescent images of 200‐cell‐stage embryos from the indicated strains stained by both anti‐SEPA‐1 and anti‐LGG‐1 antibodies. Insets show magnified views. Arrows indicate overlapping SEPA‐1 and LGG‐1 puncta. The wild type contains no visible SEPA‐1 aggregates at this stage and is therefore not shown. Scale bars: 5 μm. L The percentage of SEPA‐1 puncta co‐localizing with LGG‐1 in each strain. At least 10 embryos were scored per strain. Data are shown as mean ± SD. **P 0.0001; N.S.: not statistically different.