EMBO/sd-character-level-ner huggingface dataset:
Figure 6(A) U2OS (EGFP-Flex1-BIR-5086) cells were infected by lentiviruses expressing FANCM shRNA or shRNA vector (Ctrl) (left) or cells from the same reporter cell line expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were infected by FANCM shRNA lentiviral viruses (right). The percentage of EGFP positive cells after spontaneous recombination was quantified by FACS analysis 5 days after lentiviral infection of FANCM shRNA. Knock down of FANCM by shRNA is shown by qPCR and Western blot in Appendix Fig S12A. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01. (B) U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector were infected by lentivirus expressing FANCM shRNA. The percentage of EGFP positive cells was quantified by FACS analysis 5 days after FANCM shRNA lentiviral infection. Knock down of FANCM by shRNA is shown by qPCR and Western blot in Appendix Fig S12B. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01. (C) Proposed model for concerted roles of FANCM-dependent fork reversal and PIF1/POLD3-dependent BIR in protection of Flex1 stability. Pink arrow: endonuclease cleavage to remove Flex1. (D) Growth curves of WT or FANCM-KO HCT116 cells were plotted after expressing POLD3 (left) or PIF1 (right) shRNA or shRNA vector (Ctrl). Cell number was normalized to that on day one. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.
Figure 3A Western blot (WB) of protein lysates of HEK293 cells 72 hours post-transfection (n=3; biological replicates) with either Npu inteins or Heterologous (N-intein DnaB + C-intein DnaE) split-inteins. I + II, N6 split-intein proteins; I+II (Het.), heterologous split-intein proteins; I, 5'N6 coding sequence (CDS)-N-DnaE protein; I (N-int B), 5'N6 CDS-N-DnaB. II, C-DnaE-3'N6 CDS protein. Excised inteins (∼12kDa) are present only in the down part of the blot when I + II (Npu inteins) are provided. Arrows indicate the full-length N6 protein, single halves and excised inteins. B WB of medium of the transfected cells showing the secreted proteins (n=3; biological replicates). Arrows indicate the full-length N6 protein as well as single halves and excised inteins. C Chromogenic assay performed on the medium of transfected cells to detect F8 activity levels reported as International Units/deciliter (IU/dl). Data are presented as mean ±SEM. Each dot within each group represent a different biological replicate (n): I + II n=3; I+II (Het.) n=3; I (N-int E) n=3; I (N-int B) n=3; II (C-int E) n=3; Neg n=4. Significant differences between groups were assessed using Kruskal-Wallis rank sum test Kruskal−Wallis, p value = 0.013. *indicates the significant difference between the I+II and the I (N-int E) groups: p value ≤ 0.05. ***indicates the significant difference between the I+II and the Neg groups: p value ≤0.001.
Figure 4A, HE staining of livers with grade 0 - 3 steatosis. Scale bar: 100 µm. B, Quantification of linear unmixing of lipid in liver. Each dot represents data from one animal (grade 0: n = 14; grade 1: n = 11; grade 2 & 3: n = 9). Data represent the mean (+/- 95% confidence). The Mann-Whitney test was used to verify the statistical significance. Grade 0 vs grade 3: P = 1.22E-06; grade 1 vs grade 3: 1.19E-05; grade 2 vs grade 3 P = 0.0012 Data information: In the figure, A.U. = arbitrary units. C, Quantification of difference analysis readouts in liver. Same subjects as in B. Each dot represents data from one animal (grade 0: n = 14; grade 1: n = 11; grade 2 & 3: n = 9). Data represent the mean (+/- 95% confidence). The unpaired t test was used to verify the statistical significance. Grade 0 vs grade 1: P = 9.93E-05; grade 0 vs grade 2: P = 0.0004; grade 0 vs grade 3: P = 4.90E-05 Data information: In the figure, A.U. = arbitrary units. D, Diagnostic accuracy of linear unmixing and difference analysis in diagnosing dichotomized grades of steatosis. AUROC was calculated from data points in panel B and C. The z test was used for comparisons of AUROC between 2 groups. Grade 2 linear vs difference: P = 0.008 Data information: In the figure, A.U. = arbitrary units. E, Reconstructed MSOT image (800 nm) with linear unmixing data of Hb, HbO2, and lipid from chow and HFD-fed mice. Unmixing result: blue for Hb, red for HbO2, yellow for lipid. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value chow 1 month/ chow 2 month/ chow 3 month/HFD 1 month/HFD 2 month/HFD 3 month: Hb: 2.5/1.9/2.9/2.7/1.8/1.3; HbO2: 2.8/2.6/2.8/3.1/2.1/1.9; Lipid: 27000/19000/29000/ 32000/26000/27000). Scale bar: 4 mm. F, Longitudinal track of lipid in mouse livers. Each line represents data from one animal. Each data point is from a single measurement of each animal at each time point. All animals have grade 3 steatosis at the endpoint confirmed by histology.
Figure 3(A, B, C, D, E) Dot and box-plot comparing each of the p-tau biomarker between the Aβ negative (A-; blue; n = 287) and the Aβ positive (A+; red; n = 42) groups. Aβ positivity was defined with Aβ PET visual read. (F, G, H, I, J) Scatter plots depicting the changes between each p-tau biomarker as a function of Aβ PET Centiloids (CL). The standardized regression coefficients (β) and the P-values were computed using a linear model adjusting for age and sex. The solid lines indicate the regression line and the 95% confidence intervals. The dashed green lines indicate the CL12 and CL30 cutoffs. Participants were also colour-coded based on the CSF Aβ42/40 ratio (A-, black; A+, turquoise). (K, L, M, N, O) Dot and box-plots depicting comparison between each of the p-tau biomarker between Centiloid scale groups: (i) ≤12CL (blue; n = 278), (ii) 12 - 30CL (subthreshold Aβ pathology group; grey; n = 28), (iii) >30CL (red; n = 25). The box-plots depict the median (horizontal bar), interquartile range (IQR, hinges) and 1.5 x IQR (whiskers).
Figure 7(A) We assessed samples of cerebellar cortex, deep cerebellar nuclei, frontal cortex, pons and pyramidal tract by immunohistochemistry with ataxin-3 staining, comparing heterozygous 304Q SCA3 mice at 2, 6, 12 and 18 months of age with wildtype animals at 18 months. ML: molecular layer, PC: Purkinje cells, GL: granular layer, scale bars: 20 µm. (B) We histologically assessed the number and integrity of Purkinje cells in the cerebellar cortex by NfL, pNfH, Nissl and calbindin staining, comparing heterozygous 304Q SCA3 mice at 2, 6, 12 and 18 months of age with wildtype animals.
a) Mice with the indicated genotype were kept on a high-fat diet (HFD) for 12 weeks when indicated.figf5(a) Mice with the indicated genotype were kept on a high-fat diet (HFD) for 12 weeks when indicated. Gross liver morphology (upper panel), haematoxylin-eosin (H/E) (middle panel), and oil red O staining of liver sections (bottom panel). Ctrl, control. (b,c) Bar graph showing normalized liver weights (mean ± s.d. for n = 10 mice per group; b) and total lipid content in mice with indicated genotype (mean ± s.d. for n = 10; c). *P≤0.05;***P≤0.001 compared with control. (d) Toluidin blue staining of liver sections isolated from Atg7-LiKO mice injected with HDAd-Ctrl or HDAd-TFEB vector. (e,f) Bar graph showing normalized liver weights (mean ± s.d. for n = 10 mice per group ***P≤0.001; e) and total lipid content in mice with the indicated genotype (mean ± s.d. for n = 5 mice per group; ***P≤0.001; NS, not significant; f). Mice injected with an empty HDAd virus behaved as wild-type untreated mice; therefore, data are not represented in the figure.
Trf1 deficiency impairs K-Ras-mediated lung cancer development.Figure 2Tumor growth curve of Trf1+/+K-Ras+/G12Vp53+/+ and Trf1Δ/ΔK-Ras+/G12Vp53+/+ measured by computed tomography (CT).Quantification of the number and size of Trf1+/+K-Ras+/G12Vp53+/+ and Trf1Δ/ΔK-Ras+/G12Vp53+/+carcinomas at death point. Percentage of tumors that have deleted Trf1 quantified by TRF1 immunofluorescence after mice had been sacrificed. Post-mortem analysis of Trf1 deletion in each tumor revealed that none of the Trf1lox/lox K-Ras+/G12V p53+/+ ones had excised Trf1. Tumor growth curve and tumor growth slope of Trf1+/+ K-Ras+/G12V p53−/− and Trf1−/− K-Ras+/G12V p53−/− measured by CT. Tumor maximal section of Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−lungs measured by histological analysis before death point by CT. Maximum 18F-FDG-glucose uptake by Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−tumors 22 weeks after infection by positron emission tomography (PET).Representative PET-CT image of Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−lungs. Survival curve of Trf1+/+ K-Ras+/G12V p53−/− and Trf1−/− K-Ras+/G12V p53−/− mice.
Figure 3A Plants expressing LUC fused with full-length DOG1 (pDOG1LUC::DOG1) were sprayed with mock solution or ABA 10, 20 and 40 days after germination and analysed 24 h after treatment. The graphs show mean emitted light intensity per plant. B Representative picture of mock- and ABA-treated pDOG1LUC::DOG1 plants. The full picture is shown in Fig EV2C and D. Scale bar, 1 cm. C A truncated construct that lacks the DOG1 antisense promoter region (pDOG1shDOG1::LUC) is not induced by ABA and is highly expressed throughout development. The graphs show mean emitted light intensity per plant. D Representative picture of mock- and ABA-treated pDOG1shDOG1::LUC plants. Scale bar: 1cm. E RT-qPCR analysis of DOG1 sense mRNA level. F Strand-specific RT-qPCR analysis of antisense levels after 10 h of ABA treatment in 10-, 20- and 40-day-old Col-0 plants. G The pASDOG1::LUC reporter is silenced by the application of ABA, while mutation of TATA elements in the asDOG1 promoter (pASDOG1∆TATA::LUC) leads to attenuation of expression in the presence and absence of ABA. H Mutation of TATA elements in the asDOG1 promoter leads to high-level DOG1 sense expression and non-responsiveness to ABA. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.
AICAR but not metformin prevents IFNγ/TNFα-induced myotube atrophy.Figure 1 (A) Western blotting for phospho-Thr172-AMPK (pAMPK), total AMPK, phospho-Ser79-ACC (pACC), and total ACC 24h after treatment. Quantification represents the pAMPK/AMPK and pACC/ACC ratios relative to the non-treated (NT) control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls  (B) Phase contrast images of fibers 72h after treatment. Scale bars represent 0.25mm  (C) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls  (D-E) RT-qPCR analysis of the mRNA levels of MyoD (D 24h after treatment relative to the NT control. Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls  (D-E) RT-qPCR analysis of the mRNA levels o myogenin (E) 24h after treatment relative to the NT control. Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls
Cytoplasmic circSLC8A1 binds Ago2 and regulates miR-128 targets (A) Gene structure and conservation of the human and murine SLC8A1 gene.Figure 6(A) Gene structure and conservation of the human and murine SLC8A1 gene. The box marks the second exon that circularizes to create circSLC8A1. (B) qPCR quantification from nuclear/cytoplasmic fractionations of SH-SY cells, T-test p=0.005 for circSLC8A1, p=0.009 for NEAT1 and p=0.009 for Rpl19. n=3 biological replicas for each condition. (C) qPCR quantification of Ago2-bound RNA, immunoprecipitated (IP)/Input values normalized to negative control for a known Ago2 target, circCDR1as and circSLC8A1, T-test p=0.029 for circSLC8A1 and p=0.001 for circCDR1as, n=3 biological replicas for each condition. (D) schematic representation of circSLC8A1 with Ago2 binding sites from CLIP-seq and predicted miRNA binding sites. (E) miRNAs predicted to bind circSLC8A1 with 3 sites and more. (F) Schematic representation of circSLC8A1-Ago2 complex binding and its effect on miR-128 targets. (G) relative expression of known miR-128 targets from SN RNA-seq, p**=0.0004 for BMI1, p*=0.011 for SIRT1 and p*=0.005 for AXIN1. (H) relative expression of known miR-128 targets in SH-SY cells treated with Paraquat, T-test p*=0.03, p*=0.02 and p*=0.002 for AXIN1, BM1 and SIRT1 respectively, Data presented as mean ± SD with 4-6 biological replicas for each condition. (I) miR enrichment analysis of DE genes in SN from validated miR-targets.
A WDDEW sequence in the SNX18 LC region mediates the interaction with LC3/GABARAP.Figure 7.(A) GFP-p62 or -SNX18 full-length, SH3-LC, or PX-BAR regions were in vitro translated and incubated with GST-LC3B or -GABARAP. The resulting pulldowns were separated by SDS-PAGE. Bound proteins were detected by autoradiography and GST proteins by Coomassie blue staining. (B) 18-mer peptides covering the entire sequence of the SNX18 SH3-LC region were spotted on a membrane that was incubated with GST or GST-GABARAP, which were detected by immunoblotting against GST. The peptide sequences that specifically bound GST-GABARAP are shown with the common WDDEW motif in bold. (C) Peptides with the sequence YGGYQASQGSDDDWDDEWDDSSTVADEPGAL (SNX18 WT) or with the first (SNX18 W154S), the second (SNX18 W158S), or both (SNX18 W154S/W158S) W mutated to S were spotted on membranes that were incubated with GST, GST-LC3B, or GST-GABARAP. Binding was analyzed as in B. (D) GFP-SNX18 WT and the indicated mutants were in vitro translated and incubated with GST-LC3B or -GABARAP, and their binding was analyzed as in A. (E) HeLa GFP-LC3 cells were transfected with myc-SNX18 W154S/W158S mutant, starved for 2 h, immunostained against myc, and analyzed by confocal imaging. Bar, 10 µm. (F) Indicated myc-SNX18 constructs were transfected into HEK GFP-LC3 cells, and the number of GFP-LC3 spots per cell was quantified. The graph shows mean ± SEM (error bars), n = 3. *, P < 0.05.
Evaluation of LV.Figure 4Schematic of CD11b.IL1RN.WPRE lentivirus produced to drive codon optimised human IL1RN (secreted isoform). The biological activity of LV-mediated IL-1Ra was assessed in vitro. RAW264.7 macrophages were transduced with LV.IL1RN or LV.GFP vectors at a MOI of 10. After 72 hours cells were stimulated with recombinant hIL-1β and the expression of TNF-α assessed after a further 48 hours. (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by two-way ANOVA with Bonferroni's post-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus NTC within a treatment group. Donor MPSIIIA bone marrow was lineage depleted and transduced with LV.IL1RN vector at a MOI of 10. 3x105 transduced HSCs were transplanted into 6-8 week old busulfan myeloablated recipient MPSIIIA mice. Chimerism was assessed 2 months post-transplant and behavioural assessment conducted 4 months post-transplant. Animals were sacrificed and biochemical or histological analysis was carried out after behavioural assessment was finished. A portion of transduced HSCs were plated into CFU assays, incubated for 12 days and CFU colonies analysed. Vector copy number were quantified (n=6 CFU assays). A portion of transduced HSCs were plated into CFU assays, incubated for 12 days and CFU colonies analysed. protein production of secreted human IL-1Ra were quantified (n=6 CFU assays). A portion of transduced HSCs were plated into CFU assays, incubated for 12 days and CFU colonies analysed. intracellular human IL-1Ra were quantified (n=6 CFU assays). Donor chimerism was determined by flow cytometry at 4 months of age (2 months post-transplant) (n=10 mice). based on peripheral blood composition was determined by flow cytometry at 4 months of age (2 months post-transplant) (n=10 mice). Human IL-1Ra expression was quantified in the plasma of mice at 6 months of age (4 months post-transplant) (n=10 mice per group). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; WT vs. LV.IL1RN P<0.0001; MPSIIIA vs. LV.IL1RN P<0.0001. Symbols above bars are versus WT. Vector copy number was evaluated in brain, spleen, liver, white blood cells (WBC) and bone marrow (BM) of mice at 6 months of age (4 months post-transplant) (n=10 mice per group). Quantification of human IL-1Ra levels in the brain of transplanted MPSIIIA mice (n=10 mice per group). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; WT vs. LV.IL1RN P<0.0001; MPSIIIA vs. LV.IL1RN P<0.0001. Symbols above bars are versus WT. Representative sections from 6-month old MPSIIIA and LV.IL1RN transplanted MPSIIIA mice. Sections show the CA3 region of the hippocampus. Cells expressing high levels of human IL-1Ra are indicated with black arrows. 40x, scale bar, 50 μm (n=4 mice per group).
| NTRAS is essential for normal endothelial cell function and vital in vivo.Figure 1(A) Murine Ntras enrichment in endothelial cells versus other heart-derived cell types (data acquired from GSE95755 (n = 4 mice). Data information: In (A, data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (A) One-way ANOVA (B) Changes in lncRNA expression from HUVECs exposed to hypoxia (0.2 % O2 for 12 h or 24 h), determined by ribo-minus RNA deep sequencing (data acquired from GSE107033 ; NTRAS is highlighted (n = 2 independent biological replicates). (C) Validation of hypoxia-induced NTRAS expression at the indicated time points by RT-qPCR (n = 4 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. two-tailed unpaired t-test (D) Subcellular localization of NTRAS and control transcripts, assayed by nuclear-cytoplasmic fractionation of HUVECs and RT-qPCR (n = 3 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. two-tailed unpaired t-test (E) FITC-dextran-based in vitro permeability comparing control and NTRAS-silenced HUVECs (n = 5 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. two-tailed unpaired t-test, (F) Cumulative in vitro sprout lengths of control and NTRAS-silenced HUVECs under basal conditions and VEGFA stimulation (n = 3-10 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (G) Cell cycle analysis in Ntras-silenced H5V cells (n = 3 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (G) two-way ANOVA, (H) TMR-dextran-based assessment of vascular permeability in vivo, comparing heart homogenates from control and Ntras-silenced mice. Data normalized to organ and body weight (n = 11-12 mice per group). Experimental outline on the left. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. H) two-tailed unpaired t-test (I) Survival of control and Ntras-silenced mice under basal conditions (black, n = 21-23 mice per group) and in hind limb ischemia (HLI; red, n = 8 mice per group). Number of survivors is indicated; experimental outline on the left. Data information: (I) Mantel-Cox-test.
Figure 7A Analysis of ubiquitin conjugation to wildtype (WT) TSC1 and the indicated TSC1 mutants in 293T cells transiently transfected Flag-tagged TSC1 and its mutants along with (+) or without (-) the indicated expression vectors. B Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in lysates of TSC1-KO MEFs transfected with (+) or without (-) TSC1 wildtype or K30A expression vectors. The cells were serum starved and then left either untreated (NT) or stimulated with 10% FBS for 30 mins. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC2 (right panel) protein bands relative to the level of β-Actin. C Analysis of TSC2 ubiquitination in 293 cells transfected (for 48 h) with (+) or without (-) the indicated expression vectors. D Co-immunoprecipitation analysis of the interaction between endogenous TSC2 and exogenous TSC1 wildtype and K30A in TSC1-KO MEFs transfected (for 48 h) with (+) or without (-) the indicated expression vectors. KO MEF cells were transfected with indicated expression plasmids. The right panel is a summary graph of TSC2/TSC1 ratio based on quantification of the last four lanes of the anti-Flag IP blot in left panel. E Analysis of ubiquitin conjugation to endogenous TSC2 in TSC1-KO MEFs transfected (for 48 h) with (+) or without (-) TSC1 WT or K30A and treated with MG132 for 2 h before lysis. F Immunoblot analysis of endogenous TSC2 in TSC1-KO MEFs transfected (for 48 h) with Myc-TSC1 wildtype or K30A and subsequently treated with cycloheximide (10 μg/ml) for the indicated time periods before lysis. The right panel is a summary graph of quantified TSC2 WT and K30A protein bands presented as percentages of the level in the untreated (0 min) lane.