EMBO/sd-character-level-ner huggingface dataset:
Figure 5A, B PC9 cells (3X106/mouse) were subcutaneously injected in the flanks of CD1-nu/nu mice. When tumors reached a volume of approximately 500 mm3, mice were treated daily with erlotinib (50 mg/kg, blue area; panel A) or with osimertinib (5 mg/kg, grey area; panel B) using oral gavage. Initially, all tumors displayed stable disease or they regressed, but eventually all started relapsing. Once relapsing tumors reached 800 mm3, while under erlotinib or osimertinib treatment, we supplemented the treatment with a combination of 2XmAbs (cetuximab and trastuzumab, each at 0.1 mg/mouse/injection; twice a week) plus either erlotinib (50 mg/kg; A, red area) or osimertinib (5 mg/kg; B, orange area). Tumor volumes of individual mice are shown.
Apg9p does not comigrate with typical endomembrane markers in sucrose density gradients.Figure 4Apg9p does not comigrate with typical endomembrane markers in sucrose density gradients. A and B, Strain STY1 (pep4Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, loaded on a sucrose density gradient ranging from 18-55% (wt/wt), and centrifuged for 2.5 h at 174,000 g as described in Materials and Methods. Fractions were collected from the top (fraction number 1) to the bottom (fraction number 16). Fractions were subjected to SDS-PAGE and immunoblot with antiserum against Apg9p and: in A, Kex2p (trans Golgi network), Pho8p (vacuole), and Pma1p (plasma membrane); and in B, Sec12p (ER) and Pep12p (endosome). The graphs in panels A and B are from the same gradient, but are presented separately for clarity.
a) Homogenates from cerebral cortical tissue of a diagnosed AD case.f10(a) Homogenates from cerebral cortical tissue of a diagnosed AD case were used for the co-immunoprecipitation of human NDP52 (hNDP52) using a Tau5, 12E8 or PHF1 antibody. (b) Homogenates used for co-immunoprecipitation in a were analysed by immunoblotting using anti-NDP52, Tau5, 12E8 or PHF1 antibody. (c) Cerebral cortical tissues from diagnosed AD cases were separated into sarkosyl-soluble and insoluble fractions. The levels of total tau and tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 in the sarkosyl-insoluble fractions were analysed by immunoblotting using 12E8 and PHF1 antibodies, respectively. Samples were also immunoblotted with a polyclonal to total tau (Tau) antibody. The levels of hNDP52, p62/SQSTM1 and LC3 in the sarkosyl insoluble fractions also were examined by immunoblotting. The relative molecular masses (kDa) are indicated to the left of each blot. (d-f) The regression graphs show the relative amount of tau phosphorylated at Ser262/Ser356 (12E8) (d), at Ser396/Ser404 (PHF1) (e) or total tau (f) compared to hNDP52 present in the sarkosyl-insoluble fractions.
Figure 3(A) 786-0 or 769-P cells were pretreated with or without aa starvation for 6 hours, and then MG132 (10 μM) or CQ (1 μM) was added to the cell culture medium for 12 hours. The protein level of GDH1 was determined by WB with the indicated antibody. (B) In 786-0 cells, FLAG-tagged GDH1 was co-transfected with or without HA-tagged ubiquitin for 48 hours, and then the cells were treated with or without aa starvation for 6 hours. Immunoprecipitation was performed using an antibody against FLAG followed by western blotting using the indicated antibodies. (C) In 786-0 cells, FLAG-tagged GDH1 was co-transfected with HA-tagged ubiquitin for 48 hours, and then the cells were treated with or without aa starvation for 6 hours. Immunoprecipitation (IP) was performed using an antibody against FLAG, and the ubiquitination of FLAG-GDH1 was identified by LC-MS/MS. (D) FLAG-tagged GDH1 (including WT and mutants with different lysine mutations to arginine) was co-transfected with HA-tagged ubiquitin for 48 hours, and then cells were subjected to aa starvation for 12 hours before collecting cells. WB and qPCR separately tested the levels of GDH1 protein and mRNA, respectively. Data information: The data are represented as the mean ± SEM of three independent experiments. The one-way ANOVA test (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001). (E) FLAG-tagged GDH1 (including WT and 3KR mutant) and HA-tagged ubiquitin-expressing 786-0 cells were pretreated with aa starvation for 12 hours before treatment with MG132 (10 μM) for 12 hours. Immunoprecipitation was performed using an antibody against FLAG followed by WB using the indicated antibodies. 3KR mutant: lysine 183, 191, 200 to arginine 183, 191, 200. (F) FLAG-tagged GDH1 (including WT and 3KR mutant) was transfected into HEK293T cells and enriched using an antibody against FLAG. GDH1 enzymatic activity was quantified, and the quantity of WT and 3KR GDH1 was evaluated by CBB staining. Unpaired two-tailed t-test was used. Data information: The data are represented as the mean ± SEM of three independent experiments. The unpaired t-test were used (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001). (G) Restored expression of wild-type or 3KR mutant in 786-0 cells lacking GDH1 followed by pretreatment of the cells with or without aa starvation for 24 hours. Cells were collected, and total RNA was extracted for further qPCR using primers against the mRNA of RP genes. (H) 786-0 or 769-P cells stably expressing shGDH1 were reconstituted with rGDH1 WT or 3KR mutant and then treated with or without aa starvation for the indicated time. Cell viability was determined. Unpaired two-tailed t-test was used. Data information: The data are represented as the mean ± SEM of three independent experiments. The unpaired t-test were used (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).
Figure 5Volcano plot representing the mass spectrometry signals from 71 glycerophospholipids, namely 49 PC (phosphatidylcholine), (39 non-plasmalogens and 10 plasmalogens), 14 PE (phosphatidylethanolamine), 5 PS (phosphatidylserine), and 3 PA (phosphatidic acid), which are shown as different color symbols as indicated, analyzed in gradient-purified mitochondrial extracts from subject vs. control cells. All lipid entities shown above the horizontal red line have a p-value < 0.05.
Knocking down NEETs causes mitochondrial fragmentationA, Western blot analysis of MitoNEET expression in HeLa cells transfected with CISD1 siRNA; Bar Chart (right panel) shows replicates quantification of MitoNEET expression.Figure 2A, Western blot analysis of MitoNEET expression in HeLa cells transfected with CISD1 siRNA; Bar Chart (right panel) shows replicates quantification of MitoNEET expression. Errors bars show the standard deviation (SD) of 3 independent experiments. B, Western blot analysis of NAF-1 expression in HeLa cells transfected with CISD2 siRNA; graph (right panel) shows the replicates quantification of NAF-1 expression. Errors bars show the standard deviation (SD) of 3 independent experiments. C, RT-qPCR analysis of CISD3 mRNA levels from HeLa cells transfected with siRNA targeting CISD3. Errors bars show the standard deviation (SD) of 9 independent experiments. D, HeLa cells transfected with siRNA to reduce expression of MitoNEET (CISD1), NAF-1 (CISD2) or MiNT (CISD3) and immunostained with anti TOM20 antibody. Arrowheads indicate fragmented mitochondria. E, Quantification of mitochondria morphology (based on TOM20 immunostaining as shown in D) from HeLa cells transfected with siRNA targeting mitoNEET (siCISD1), NAF1 (siCISD2) or MiNT (siCISD3). Quantification is expressed as ratio of tubular and fragmented mitochondria. Errors bars show the standard error of the mean (SEM). (n =125-130 cells). F, Electron microscopy pictures from 150 INS-1 cells transfected as described in G. G, Western blot analysis of NAF-1 expression in cells stably transfected with control shRNA, shRNA targeting reduction of NAF-1 protein expression (shCISD2), or shRNA targeting reduction of NAF-1 protein expression complemented with plasmid derived expression of NAF-1 (shCISD2+CISD2); western blot quantification of NAF-1 is shown on the accompanying bar chart. Errors bars show the standard deviation (SD) of 3 independent experiments. H, Quantification of mitochondrial length in EM images from cells transfected as described in G. Errors bars show the standard error of the mean (SEM) (n=260-270, technical replicates).
Figure 7ATM-proficient cells respond to DSBs with initial, robust ATM-mediated phosphorylation of numerous substrates, after which ATM's activity is attenuated in a DNA-PK-dependent manner. In ATM-deficient cells, some ATM substrates are targeted by DNA-PK at the early phase of the response, or by ATR in the later phase, but to a lesser extent than in WT cells. ATR-dependent compensation for ATM absence in A-T cells includes significantly more substrates than that of DNA-PK and involves sites that ATR usually targets in response to genotoxic stresses other than DSBs. A few of these can be targeted by ATR also when ATM is chemically inhibited in WT cells.
Figure 2(A) Table shows the total number of pups, the number or frequency of human HC-, human LC-and human BCR- KI pups after One-step CRISPR/Cas9 microinjection of CLK21. (B) CLK21 KI mice F0 and F1 generations. Squares represent male mice and circles represent female mice. Upper halves of squares or circles represent Ig κ, and the lower halves represent Ig H, as shown in the schematic. F0 generation mice genotyping results showing 7, 9 and 14 are HCLK21/WTκWT/WT, 1 and 4 are HWT/WTκCLK21/WT, and 2, 3, 6, and 12 are HCLK21/WTκCLK21/WT. Mouse 3 was crossed with WT to obtain 11 F1 progeny: Five F1 mice are HWT/WT κCLK21/WT, four are HCLK21/WTκCLK21/WT and two are WT. (C) Single cell sequencing for naïve B cells from heterozygous CLK21 double KI mice. Left column shows the gating strategy for sorting naïve B cells (upper) and eOD-GT8 specific naïve B cells (lower). Right pie charts show the frequency of paired HC and LC sequences among total naïve B cells (upper) and eOD-GT8 specific naïve B cells (lower). (D) Germline-targeting eOD-GT8 binding activity of B cells from WT, HCLK21/WTκWT/WT, HWT/WT κCLK21/WT), and HCLK21/WT, κCLK21/WT KI mice. 8-week-old mice were detected by FACS. X and Y axes represent that BCR were stained with eOD-GT8 tetramer conjugated with Alexa Fluor™ 488 and Alexa Fluor™ 647 respectively. Representative dots were gated from Scatter/Singlet/Live (SSL), B220+ IgM+ IgD+. (E) Quantification of eOD-GT8 binding in CLK21 KI mice. X axis represents F0 (n=1) and F1 (n=3) KI animals, and the Y axis represents the percentage of eOD-GT8 targeting binders in mature (IgM+IgD+) B cells. Bars indicate mean ± SD from mice in each group.
Figure 4(A) Percentage of change in field excitatory postsynaptic potential (fEPSP) recorded after theta-burst stimulation (TBS) in Schaffer collaterals of hippocampal slices from 6 month old wild type, 5xFAD and 5xFAD/p75NTR mutant mice, as indicated. Results are presented as mean % change normalized to t=0 ± SEM. N=6 (wild type), 5 (5xFAD), 9 (5xFAD/KO), 7 (5xFAD/ΔDD) and 8 (5xFAD/C259A) slices from 3 mice per genotype, respectively. (B) Quantification of fEPSP (mean % change ± SEM) in the indicated genotypes at 3 time points. *, P<0.05; **, P<0.01 vs. 5xFAD (two-way ANOVA followed by post hoc test). N numbers as in (A). (C) Behavior in the novel object recognition (NOR) test of 6 month old wild type, 5XFAD and 5XFAD/p75NTR mutant mice, as indicated. Histograms show mean recognition index ± SEM during training, and 30min, 24h and 14 days after training, corresponding to measures of short term, long term and remote memory, respectively. Bar color codes are as in panel (A). *, P<0.05 vs. 5xFAD (two-way ANOVA followed by post hoc test). N=12 (wild type, 5xFAD/ΔDD and 5xFAD/C259A), 10 (5xFAD) and 8 (5xFAD/KO) mice per genotype, respectively. (D) Training latency in the Barnes maze test of 6 month old wild type, 5XFAD and 5XFAD/p75NTR mutant mice, as indicated. Histograms show mean latency in seconds to find the platform hole ± SEM in 4 consecutive training sessions. Bar color codes are as in panel (A). *, P<0.05 vs. 5xFAD; #, P<0.05 vs. wild type, 5xFAD/ΔDD or 5xFAD/C259A (two-way ANOVA followed by post hoc test). N=14 (wild type and 5xFAD/ΔDD), 10 (5xFAD, (5xFAD/KO and 5xFAD/C259A) mice per genotype, respectively. N=14 (wild type and 5xFAD/ΔDD), 10 (5xFAD, (5xFAD/KO and 5xFAD/C259A) mice per genotype, respectively. (E) Percentage of time spent (mean ± SEM) in the target quadrant of the Barnes maze test 30min, 24h and 14 days after training. *, P<0.05 vs. 5xFAD; #, P<0.05 vs. wild type, 5xFAD/ΔDD or 5xFAD/C259A (two way ANOVA followed by post hoc test).
Palmitoylated Golga7b stabilises endogenous DHHC5 at the plasma membrane.Figure 3Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG-tagged WT Golga7b. Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG-tagged mutant Golga7b. Confocal images of endogenous Golga7b and DHHC5 in HeLa cells treated with negative control non-targeting siRNA. Confocal images of endogenous Golga7b and DHHC5 in HeLa cell treated with Golga7b siRNA. Quantification of plasma membrane signal of endogenous DHHC5 when co-expressed with WT or mutant Golga7b or without any transfected Golga7b. ****=P<0.001, t-test, endogenous + WT Golga7b n=37, endogenous + mutant Golga7b n=23, endogenous n=42. Expression of the palmitoylation deficient mutant Golga7b causes endogenous DHHC5 to be internalised in the cell, while WT Golga7b stabilises DHHC5 at the plasma membrane. Quantification of the plasma membrane signal of DHHC5 in (C and D), ****=p<0.001 t-test, control siRNA n=23, Golga7b siRNA n=23.
Plasmin cleaves VN in the RGD-motif.Figure 2A Pre-treatment of VN with plasmin, but not uPA, inhibits subsequent cell adhesion mediated by uPAR and integrins. E-Plates wells coated with VN were incubated with a dilution curve of plasmin or tc-uPA for 1 h at 37˚C prior to cell seeding. After washing, 293/uPARWT or HeLa cells were seeded and their adhesion was followed by RTCA. The cell indexes recorded 1 h after seeding are shown. Data are reported as percentage of the cell index measured in non-pre-treated wells and represent the mean ± SD of three independent experiments. B Pre-treatment of FN with plasmin or tc-uPA does not affect subsequent cell adhesion. RTCA experiments were performed as described for panel A, but with FN-coated wells. E-Plates wells coated with FN were incubated with a dilution curve of plasmin or tc-uPA for 1 h at 37˚C prior to cell seeding. After washing, 293/uPARWT cells were seeded and their adhesion was followed by RTCA. The cell indexes recorded 1 h after seeding are shown. Data are reported as percentage of the cell index measured in non-pre-treated wells and represent the mean ± SD of three independent experiments. C Plasmin cleaves VN at the 45RG peptide bond. Urea purified VN was incubated with plasmin (substrate/enzyme ratio: 10:1 w/w) for 2 h at 37°C and the reactions were analysed by MALDI-TOF mass spectrometry.
Figure 1A) Schematic illustration of Suv39h1 protein. ΔN-Suv39h1 lacks the 41 N-terminal amino acids but contains a chromodomain. B) Co-immunoprecipitation analysis to elucidate the interaction between Suv39h1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Suv39h1 or 3xFlag-ΔN-Suv39h1 were subjected to immunoprecipitation with the anti-Flag tag antibody followed by immunoblotting with the indicated antibodies. IgG represents the heavy or light chain C) Protein decay analysis of HP1 interaction-defective Suv39h1 mutant. mESC lines stably expressing 3xFlag-Suv39h1 or 3xFlag-ΔN-Suv39h1 were treated with CHX and MG132 for the indicated times. p53 was used as the positive control for the protein decay analysis. Stacked bar plots show the summarized protein amounts of Suv39h1 (bottom). Data represented as mean ± SD (n = 4, biological replicates). **p < 0.01 (Tukey's honestly significant difference test). D) Schematic illustration of Setdb1 protein with putative HP1-binding motifs. Setdb1-5A and Setdb1-3A carry substitutions of amino acids from 344PMVLL348 to AMALA and from 617PLLVPL622 to ALLAPA, respectively. E, Co-immunoprecipitation analysis to elucidate the interaction between Setdb1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Setdb1 or 3xFlag-Setdb1 mutants were subjected to immunoprecipitation with the anti-Flag tag antibody (E) The immunocomplex was analyzed by immunoblotting with the indicated antibodies. F) Co-immunoprecipitation analysis to elucidate the interaction between Setdb1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Setdb1 or 3xFlag-Setdb1 mutants were subjected to immunoprecipitation with the anti-HP1β antibody The immunocomplex was analyzed by immunoblotting with the indicated antibodies. G) Protein decay analysis of HP1 interaction-defective Setdb1 mutant. mESC lines stably expressing 3xFlag-Setdb1 or 3xFlag-Setdb1-3A were treated with CHX and MG132 for the indicated times. Stacked bar plots show the summarized protein amounts of Setdb1 (right). Data represented as mean ± SD (n = 4, biological replicates). **p < 0.01 (Tukey's honestly significant difference test).
Fig.Figure 2Four to six-week-old C57BL/6 mice were injected i.p. with 104-107 PFU of WNV-poly(A), 104 PFU of WT WNV or PBS (negative control) in a volume of 200 μL. Survival (n=5) in infected mice were monitored daily Data information: Log-rank test was used Four to six-week-old C57BL/6 mice were injected i.p. with 104-107 PFU of WNV-poly(A), 104 PFU of WT WNV or PBS (negative control) in a volume of 200 μL. body-weight changes in infected mice were monitored daily (n=5, the values represent mean ± sd in each group) Data information: two-way ANOVA analysis were used * p < 0.05, *** p < 0.001, ****p < 0.0001, ns represents not significant. Four to six-week-old C57BL/6 mice were injected i.p. with 104-107 PFU of WNV-poly(A), 104 PFU of WT WNV or PBS (negative control) in a volume of 200 μL. viremia was quantified by plaque assay from days 1 to 3 post-infection (n=5, the values represent mean ± sd in each group, each symbol represents a mouse) Data information: one-way ANOVA analysis were used * p < 0.05, *** p < 0.001, ****p < 0.0001, ns represents not significant. Four to six-week-old C57BL/6 mice were injected i.p. with 104-107 PFU of WNV-poly(A), 104 PFU of WT WNV or PBS (negative control) in a volume of 200 μL. Histological analysis of mouse brain. Scale bars represent 100 μm and 50 μm for left and right panels, respectively. Severe vacuolization (black arrows) and inflammation (yellow arrow) in brain small vessels as well as degenerative changes in neurons with cytoplasmic rarefaction (blue arrow) and necrosis with nuclear compaction (red arrow) were indicated. Scale bars represent 100 μm and 50 μm for left and right panels, respectively E Viral load and replication kinetics in different tissues of C57BL/6 mice. Six-week-old female C57BL/6 mice were inoculated subcutaneously with 106 PFU of WT WNV or WNV-poly(A) in the left rear footpad, and tissues were harvested from two mice at the indicated times post-inoculation. Viral loads in brain, spleen, left lymph node, right lymph node and liver were measured by plaque assay and expressed as PFU per gram of tissue. n=3, values are mean ± sd, the dotted line corresponds to the limit of detection (LOD).
Figure 3Schematic representation of the Shh pathway in the cilium. MECP2-silenced or control hTERT-RPE-1 cells were starved for 48 h before treatment with 200 nM and 500 nM SAG for 24 h. Cells were stained with anti-Smo and anti-acetylated α-tubulin antibodies. The percentages of Smo-positive cilia were calculated. The graph shows the average of three independent experiments (mean ± S.E., *** p<0.001, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA demonstrated a significant silencing effect (F (1, 12) = 160; p<0.0001), a significant SAG treatment (F (2, 12) = 259.4; p<0.0001) and a significant interaction (F (2, 12) = 36.85; p<0.0001). WT or Mecp2 null MEF cells were treated with SAG (200 and 500 nM) for 24 h and the percentages of Smo-positive cilia were calculated. The graph shows the average of three independent experiments (mean ± S.E., *p<0.05, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA indicated a significant genotype effect (F (1, 12) = 11.22; p<0.01) and a significant SAG treatment (F (2, 12) = 11.7; p<0.0001). Gli1 protein levels were measured by Western blot in untreated (UT) and 100 nM SAG-treated WT and Mecp2 null MEF cells (n=5/6). Representative bands of Gli1, Mecp2 and α-tubulin are depicted above the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to the untreated WT samples (**p<0.01, ***p<0.001, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA reported a significant interaction (F (1, 18) = 6.652; p<0.05), a significant genotype effect (F (1,18) = 15.95; p<0.001) and a significant treatment (F (1, 18) = 20.15; p<0.001). Gli1 protein levels were measured by Western blot in WT and Mecp2 null neurons, following SAG treatment (200 and 400 nM). Representative bands of Gli1, Mecp2 and α-tubulin are depicted above the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to the untreated WT samples (*p<0.05, ***p<0.001, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA indicated a significant interaction (F (2, 20) = 3.788; p<0.05), a significant genotype effect (F (1, 20) = 4.362; p<0.05) and a significant treatment (F (2, 20) = 13.5; p<0.001). Gli1 mRNA expression levels upon SAG exposure in MEF cells (n=7 WT and n=9 null samples) (F; SAG 100 nM) and cortical neurons (n= 15 WT and n=12 null samples) (G; SAG 400 nM). The graphs show the mean ± S.E. of the fold change derived from at least three independent experiments (mean ± S.E., **p<0.01, by two-way ANOVA, followed by Bonferroni post hoc test).
Figure 4A. Ratio of hemolymph to midgut sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. B. Ratio of salivary gland to midgut sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. Statistical analysis: (A) and (B) One-way analysis of variance with Tukeys Multiple Comparison test. C. Blood stage infection of mice after transmission by mosquito bite. Shown is the mean ± standard deviation. D. Blood stage infection of mice after i.v. injection of 1000 sporozoites.
Figure 2TEM tomograms from high-pressure frozen embryos. (A-A') Low magnification (2.5 nm voxels) tomogram and 3D reconstruction from 10 serial 300 nm sections that cover one terminal cell. (A''-A''') 3D reconstructions derived from higher resolution tomograms showing different organelles in the cell. (B) Example of an individual high-resolution (0.78nm voxels) tomograms used to generate 3D reconstructions. (C) Low resolution tomogram of another cell; the dotted box is magnified in (D). (D) High-resolution tomogram. (D') Model derived from 4 serial 3D reconstructions including the one shown in (D).
Nesprin mobility is greater than lamin A/C mobility.Figure 3A, The deformed nucleus of cells migrating through narrow constrictions was bleached at the front and the side to determine mobility. Inset are shown close-up views of the bleached areas at the front of the nucleus. B, Quantification of the recovery. Error bars correspond to the standard error of the mean, n=24 over 3 biological replicates.