EMBO/sd-character-level-ner huggingface dataset:
Figure.Figure 6Z α1-antitrypsin transgenic animals were dosed with 10, 30 or 100 mg/kg GSK716 three times a day. Total concentrations of GSK716 were determined by LC-MS/MS (Table 2). (A) 100mg/kg GSK716 resulted in a mean 7-fold increase in circulating monomeric Z α1-antitrypsin levels demonstrating robust target engagement in the liver. The observed steady state total and free compound levels of GSK716 in the transgenic Z α1-antitrypsin mouse were well-predicted by the in silico PK model built on: (1) in vitro metabolic clearance data, (2) plasma protein binding data, (3) in vivo PK data from wild type mice and (4) a term comprising a 5μM circulating sink for drug with an affinity of 1.5 nM, representing the Z α1-antitrypsin within blood. The target free drug concentration was selected based on the observed potency in the in vitro secretion assays in which the total drug approximates to the free drug in the assay. Interestingly, 30mg/kg and 10mg/kg groups also gave significant, dose-dependent increases in circulating Z α1-antitrypsin despite free concentrations being below the cellular EC50 for secretion for much or all of the dosing period. Significance at p<0.05 by a student's t test is denoted by *, shown as pairwise comparisons (each animal is compared with itself, treated vs pre-treatment) and between groups (compound treated vs vehicle), with n= 7, 8 and 8 per group for day 6 dose level 10, 30 and 100mg/Kg respectively; day 15, n=10; day 21, n=5. Data presented as mean ± SD. B) Representative example images of 2C1-stained livers from a 20-day vehicle (i and ii) and 100mg/kg TID GSK716 (iii and iv) treated animals. The asterisks indicate regions of hepatocytes that are negative on 2C1 immunostaining. Scale bar (i) and (iii) 2mm and (ii) and (iv) 100 µm. C) Quantification of 2C1 stained area in livers from vehicle and 100mg/kg TID GSK716 treated animals at day 15 (top) and 21 (bottom), shown as total area and low-only or high and mid-only intensity stained areas. There were no significant differences between groups by Mann-Whitney U test; graphs show median±interquartile ranges, with n=5 per group.
Figure 2The isoprene tail is synthesized from farnesyl pirophosphate by PDSS1/PDSS2 and condensed to the 4HB ring by COQ2. The ring in processed in CoQ by COQ3-7. ADCK3/CABC1 (COQ8) regulates CoQ biosynthesis through phosphorylation of COQ3, COQ5 and COQ7.
a, LC3 staining of representative pancreatic tumours from mice of indicated genotypes.f4a, LC3 staining of representative pancreatic tumours from mice of indicated genotypes. Scale bars: 50 µm. b, Western blotting for LC3, p53 and Atg7 in cell lines derived from individual tumours which developed in the absence or presence of p53. c, d, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR, c, d, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR, d) of eight Atg7+/+ and seven Atg7−/− cell lines revealed increased ECAR in autophagy-deficient cells. Error bars are s.e.m. e, Treatment with 2-deocyglucose (2-DG) reduces ECAR and abrogates the difference between cell lines from Atg7+/+ and Atg7−/− tumours. Values are the average of three biological replicates. Error bars are s.e.m. , g, LC-MS analysis revealed increased glucose consumption from medium (f) and extracellular lactate accumulation (g) in Atg7-null cell lines. Values are the average of eight (Atg7+/+) and seven (Atg7−/−) biological replicates. h, LC-MS analysis shows increased accumulation of 2-DG and its metabolite 2-DG-6-phosphate in Atg7-deficient cell lines. Values are averages of five biological replicates for each genotype. i, Background levels of 18F-FDG uptake in brain and heart can be seen in sagittal and coronal views (middle and right panels, respectively) of wild-type Atg7 mice (top row). In contrast, intense 18F-FDG uptake is seen in pancreatic tumours of Atg7 deficient mice (bottom row). j, LC-MS of intracellular glycolytic and pentose phosphate pathway intermediates shows enhanced accumulation in Atg7−/− tumour cell lines. Values are average of eight (Atg7+/+) and six (Atg7−/−) biological replicates. f-h, j, Error bars are s.e.m. a, f-h, j, A Mann-Whitney U-test was used to ascertain significance. *P  0.05, **P  0.01.
The UBE2R2 R149E variant disrupts acceptor Ub binding and gating loop flexibility.Figure 4A Dissociation constants of UBE2R2 and its variants binding to acceptor Ub as determined by BLI and fitted data from METRIS. Data from n≥3 independent experiments. B Schematic of METRIS assay set-up and parameter analysis to assess the binding between UBE2R2 and Ub. In brief, biotinylated Ub is linked to a magnetic bead that is introduced to a magnetic field while on a UBE2R2 functionalized coverslip. The movement of each bead is quantified, and the rolling parameter correlates to binding affinities between Ub and E2. C Plot of the METRIS rolling parameters of UBE2R2 WT, D143K, and R149E compared using one-way ANOVA (**** p ≤ 0.0001, WT: 63 beads x 17 rolls n=1071, R149E: 46 beads x 17 rolls n=782, D143K: 47 beads x 17 rolls n=799; technical replicates), indicating a weaker interaction with Ub between the UBE2R2 variants compared to WT. Error bars: SEM. D Docking model of the UBE2R2 (grey) - acceptor Ub (orange) interaction with E2-Ub contacts (blue), UBE2R2 gating loop (yellow), and R149 (purple) highlighted. Dashed line indicates distance measured for Fig 4E between position 149 and T136 (Hill et al., 2016). E UBE2R2 R149E restricts the movement of the gating loop compared to WT as shown by the distances of the Cα atoms from T136 and R149 in molecular dynamics simulations. F Plot of METRIS rolling parameters of WT and the R149E substituted variant of UBE2R2 isopeptide-linked to the donor Ub compared using one-way ANOVA (* p ≤ 0.05, WT-Ub: 47 beads x 17 rolls n=799, R149E-Ub: 114 beads x 17 rolls n=1938; technical replicates). Error bars: SEM. Role of the UBE2RHTH in diUb formation is determined from kinetic parameters (G-H) for mutants upon titrating the acceptor Ub in pulse-chase reactions following UBE2R2~*Ub Apparent Km and Vmax were determined using the Michaelis-Menten equation (n≥3 independent, technical replicates). Error bars: SEM. Data normalized to the WT apparent Vmax. Role of the UBE2RHTH in diUb formation is determined from kinetic parameters for mutants upon titrating the acceptor Ub in pulse-chase reactions following UBE2R2~*Ub monitored by fluorescent scanning of nonreducing SDS-PAGE gels
AB-Tie1-39 treatment stabilizes lung vessels to restrict extravasation of disseminated tumor cells.Figure 3(A) Mice were pretreated with IgG or AB-Tie1-39. Thereafter, B16F10 cells were intravenously injected to initiate experimental metastasis independent of the primary tumor. (B) Representative images of the whole lungs are shown on the left. Scale bars = 5 mm. Quantitation of the number of lung metastatic foci is plotted on the right (mean ± SD, nIgG = 9, nAB-Tie1-39 = 8 mice). *, P<0.05 (two-tailed Mann-Whitney U test). (C) EC were seeded on gelatin-coated Transwell inserts. RFP-labeled LLC cells were allowed to transmigrate for 8 h through the endothelial layer. Thereafter, transmigrated tumor cells were counted. The dot plot presents the normalized data (mean ± SD, n = 9 inserts from three independent experiments). ***, P<0.001 (two-tailed Mann-Whitney U test). (D) LLC tumor-bearing mice were pretreated with IgG or AB-Tie1-39. On day 14, lung tissue lysates were immunoblotted with anti-phospho-TIE2, total-TIE2 and ACTB (upper panel; n = 6 mice). Densitometric quantitation of the blots presented in the upper panel is shown (mean ± SD, n = 6 mice). **, P<0.01 (two-tailed Mann-Whitney U test). (E) LLC tumor-bearing mice were pretreated with IgG or AB-Tie1-39. On day 14, lung EC were FACS-sorted and used for global gene expression (n = 4 mice). On the left, a heatmap shows significantly- regulated genes (red outline = AB-Tie1-39-treated; black outline = IgG-treated). On the right, top significantly regulated bio-functions in AB-Tie1-39-treated as compared to IgG-treated mice in Ingenuity Pathway Analysis.
Noise-induced hearing damage.Figure 6Noise exposure and ABR measurements. Exposure of mice to broadband noise (2-10 kHz) of either 108 or 110 dB SPL resulted in elevations of auditory thresholds. Thresholds in Mrps5V338Y/V338Y mutant mice (black bars) were significantly higher after exposure than thresholds in littermates (open bars), (p<0.0001, two-way ANOVA for combined 108 and 110 dB data). Frequencies of 12, 24 and 32 kHz were selected for measurement to cover the mid-frequency to high-frequency regions of the cochlea. n=6 for baseline littermates; n=6 for baseline mutants; n=3 for all noise exposures.
Figure 1(A) Heteroplasmy levels by PCR-RFLP at position 3243 in 82 hiPSCs clones at passage 2, derived from three MELAS patients (MitoA, MitoB, MitoC). The black dots represent the mutation load for a specific hiPSC clone; n is the total number of individual hiPSC clones analyzed per patient. The red stars represent the heteroplasmy levels for the parental fibroblasts. (A) Heteroplasmy levels by PCR-RFLP at position 3243 in 82 hiPSCs clones at passage 2, derived from three MELAS patients (MitoA, MitoB, MitoC). The black dots represent the mutation load for a specific hiPSC clone; n is the total number of individual hiPSC clones analyzed per patient. The red stars represent the heteroplasmy levels for the parental fibroblasts.
Fig.Figure 6(A) Xanthomonas campestris cells treated with FeSO4, H2O2 and FeSO4+Dp were analyzed for ROS by monitoring DCF fluorescence with fluorometry. Data shown are mean ± S.E. (n = 3). (B) The percent of ROS positive Xanthomonas campestris cells treated with FeSO4, H2O2 and FeSO4+Dp determined by flow cytometry at 3 h after treatment. Shown are the mean percentages of ROS positive cells exceeding the fluorescence of 99% of untreated cells. Data shown are mean ± SE (n=3). *** indicates p-value < 0.001 compared with data obtained from iron treated wild type and mutant by paired student t-test. (C) Iron sensitivity assay. Cells were grown overnight in PS medium and cultures were diluted in fresh media at an density of 1 x 106 cells/ml, treated in the absence or presence of 500 µM FeSO4. Viability of cultures was measured at the indicated time point by dilution plating. Percent survival was determined as the number of viable cells at each time divided by the number of viable cells before exposure to FeSO4. Data shown are mean ± SE (n=3). * indicates p-value < 0.05 and ** indicates p-value < 0.01 compared with data obtained from iron treated wild type and mutant by paired student t-test.
Figure.Figure 6A. MEFs were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo for the indicated time. Cells were lysed with SDS-containing sample buffer or Triton buffer. Lysates were analyzed by immunoblotting with the indicated antibodies. B. MEFs were treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12h, or 100 μM Dfo for 6 h after treatment with 10 μg/ml FAC for 12 h. Triton-soluble lysates were subjected to glycerol gradient ultracentrifugation analysis. C. WT and TAX1BP1 KO MEFs were pretreated with 25 μg/ml FAC for 16 h and then cultured with 100 μM Dfo. Lysates were fractionated and then analyzed by immunoblotting with the indicated antibodies. D. Soluble FTL bands in (C) were quantified by densitometry. Data are shown as the means ± SD of three biological replicates. E. TAX1BP1 KO MEFs (sg1 clone) stably expressing FLAG-OsTIR1 (F74G) and FLAG-mAID-mTAX1BP1 were pretreated with 10 μg/ml FAC for 12 h, cultured with DMSO or 1 μM 5-Ph-IAA for 2 h, and then treated with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies. 5-Ph-IAA, a derivative of Auxin; mAID, mini auxin-inducible degron F. TAX1BP1 KO MEFs (clone sg1) in which ATG7 was knocked down were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies. G. TAX1BP1 KO MEFs (clone sg1) stably expressing myc-hNCOA4 pretreated with 25 μg/ml FAC for 16 h were cultured with 100 μM Dfo, and soluble lysates were analyzed by immunoblotting with the indicated antibodies. H, I. NCOA4 KO MEFs reconstituted with NCOA4 variants were pretreated with 10 μg/ml FAC for 12 h and then chased with (H) 20 μg/ml CHX or (I) 100 μM Dfo for the indicated times. Soluble lysates were analyzed by immunoblotting with the indicated antibodies. J. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 3 h (indicated as F) or 100 μM DFO for 3 h after treatment with 10 μg/ml FAC for 12 h (indicated as D), and then cultured with DMSO or 50 μM biotin for 30 min. Triton-soluble cell lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed by immunoblotting with the indicated antibodies. The black and white arrowheads indicate hNCOA4-TurboID and endogenous NCOA4, respectively.
Sema3C inhibits endothelial cell survival and disrupts endothelial cell–cell contacts.Figure 4HUVECs were treated with Sema3C- or GFP-conditioned medium for 24 h. Cell proliferation was quantified by BrdU incorporation (mean ± s.e.m; n = 3; n.s., non-significant). Apoptosis of HUVEC was measured by caspase-3 and caspase-7 activities after 3 h of incubation with Sema3C- or control-conditioned medium (mean ± s.e.m; n = 3, *P = 0.018 using unpaired Student's t-test). Serum-starved HUVECs were incubated with conditioned medium or conditioned medium + VEGF (50 ng/ml) for 15 min. Cell lysates were subjected to SDS-PAGE and immunoblotted with anti-phospho-AKT antibody.Quantification of phospho-AKT normalized to total AKT levels (mean ± s.e.m; n = 4; control vs. Sema3C (− VEGF), *P = 0.034 and control vs. Sema3C (+ VEGF), *P = 0.024 using unpaired Student's t-test). HUVECs were adenovirally transduced with VE-cadherin-GFP and plated on ibidi slides (n = 4; control vs. Sema3C (− VEGF) at 20× magnification (Supplementary Movies S1 and S2)). Representative pictures are shown at time points 0 and 1 h. Arrowheads indicate gaps between endothelial cells. Scale bar, 100 μm. C57BL/6Nmicepups at P5 received 1 μg of recombinant Sema3C or IgG-Fc proteins. Endothelial cell junctions were examined by whole-mount VE-cadherinimmunofluorescence staining 6 h later. Representative images show VE-cadherin at cell junctions (arrows). The linear structure (i) was disrupted in Sema3C-treated retinae (ii). Scale bar, 100 μm (upper panel) and 20 μm (lower panel). Whole-mount immunostaining of the vascular bed by CD31 and apoptotic cells by cleaved caspase-3 in the retinae at P5 8 h after intravitreal injection of Sema3C or IgG-Fc. Representative images show active caspase-3 signals that co-localize with endothelial cells (arrows). Scale bar, 100 μm.Quantification of cleaved caspase-3 normalized to area of CD31-stained vessels (mean ± s.e.m; n = 5; **P = 0.007 using paired Student's t-test).
a, Representative mitochondrial morphology in Smurf1+/+ (wild-type) and Smurf1−/− (KO) MEFs transfected with indicated construct and treated with DMSO or 10 &mgrM CCCP for 24 h.f4a, Representative mitochondrial morphology in Smurf1+/+ (wild-type) and Smurf1−/− (KO) MEFs transfected with indicated construct and treated with DMSO or 10 µM CCCP for 24 h. b, Quantification of percentage of total cells with a diffuse accumulation of abnormal fragmented mitochondria and lack of mitochondrial clearance. Results shown represent combined data from 3-5 experiments per condition with triplicate wells (of at least 100 cells per well) analysed for each condition per experiment. Shown are mean ± s.e.m. for average values from each experiment. Similar results were observed in each independent experiment. *P  0.001, Students t-test. c, Measurement of mitochondrial fractional area (percentage of total cellular area) in MEFs treated as in a. Results shown represent mean ± s.e.m. for 50 cells per condition. d, Representative confocal micrographs of KO MEFs transfected with YFP-SMURF1 wild-type or YFP-SMURF1Dgr;C2 (Dgr;C2) and treated for 4 h with DMSO or CCCP. e, Representative confocal micrographs of KO MEFs transfected with GFP-LC3 and wild-type mCherry-SMURF1 (WT) or mCherry-SMURF1Dgr;C2 (Dgr;C2) and treated for 4 h with CCCP. Inset, upper right, formation of completed autophagosome around a damaged mitochondrion associated with wild-type SMURF1; insets, lower right, incomplete autophagosomes or absence of LC3 signal around mitochondria associated with SMURF1Dgr;C2. See also Supplementary Figs 10 and 11 for enlarged images. f, Representative electron microscopy images of indicated tissues from 10-month-oldSmurf1+/+ (WT) or Smurf1-/- (KO) mice. Arrows, representative abnormal mitochondria. Scale bars, 1 µm. Similar abnormalities were observed throughout entire electron microscopy tissue section for each mouse and in tissue samples from three different mice for each genotype.
Figure 3A Protein expression of wild-type and KEN-mutant E2F7 and E2F8 in 293T cells in presence or absence of CDH1-flag 48 hours after transfection. For E2F8, KEN motifs starting at amino acid 5 (KEN5) or 374 (KEN374) were mutated separately into 3 consecutive alanines or in combination (K/Kmut). Blots are representative of two independent experiments. B Immunoblots showing repression of the E2F target genes CDC6 and Cyclin A in HeLa cells with stable doxycycline-inducible expression of wild-type and KEN double-mutant E2F7/8 after 16 hours of doxycycline treatment. C FACS plots showing DNA content on the x-axis (propidium iodide) of HeLa cells with stable expression of wild-type or KEN-mutant E2F7, after 24 hours of doxycycline. Thresholds for EGFP positivity were set by applying the same gate to vehicle- and doxycycline treated cells. D HeLa cells expressing inducible E2F7-EGFP (left destruction graph) or E2F8-EGFP (right destruction graph) were blocked with thymidine for 16h, then released in fresh medium with doxycycline. Imaging was performed at 3-min intervals, as in Figure 2E. The x-axis is set to 0 at the frame of anaphase onset. Graphs show mean ± s.e.m. wild-type (wt) shown from Figure 2E in gray. E2F7-EGFP-KENmut n=13 from 2 independent experiments, E2F8-EGFP K/Kmut n=26 from 3 independent experiments. E Montages of representative cells from experiments shown in (D). Time (min) indicates time from onset of anaphase.
Figure 2 A. Schematic illustration of FUS‑NTD‑GST truncation constructs. Amino acid numbers are indicated  B. Immunoblot analysis (IB) of GST-pulldown samples with deletion mutants of FUS‑NTD‑GST as baits with/without cell extract (C.E). ARID1A and BRG1 were used as tracers of SWI/SNF binding. Coomassie staining of recombinant proteins are shown in the lower panel. Amino acid numbers are indicated  C. Immunoblot analysis (IB) of FUS-NTD‑GST pulldown samples of cell extracts (C.E.) after treatment with RNase or DNase, using antibodies against ARID1A and BRG1  D. Immunoblot analysis (IB) of SWI/SNF components (ARID1A, BRG1, BAF170, BAF155, BAF60A, SS18 and BAF47) and full-length FET proteins (EWSR1 and FUS) remaining on sepharose-bound FUS-NTD‑GST pulldown baits after increasing stringency washes with 100, 150, 250, 500 or 1000 mM NaCl. Note loss of BRG1 at NaCl concentration > 250mM
Figure 3Two-tier analysis strategy and design for the confirmation of processing areas in Alu RNAs in human brain combining different sequencing approaches and patient cohorts (CBB cohort , n=24; MAP cohort , n=241). Plotting of the position of the 5'end of Alu RNA fragments across the Alu metagene to depict potential processing areas of Alu RNAs in all post-mortem cortex tissues from patients from the MAP cohort. As in Figure 1B each row represents the distribution of normalized counts of the 5'ends of reads mapped across the Alu metagene for each patient. As in Figure 1B x-axis numbers represent the distance from the Transcriptional Start Site (TSS) area and heatmap density corresponds to normalized counts of the 5'end of the reads with red for higher density of these 5'ends at a specific position. XR1 as in Figure 1B.
IEC-specific loss of OGT disrupts intestinal proliferative/apoptotic homeostasis.Figure 3 A Representative images of the H&E staining of ileum and colon of 10-week-old male wildtype and Vil-Ogt KO mice. Scale bars = 100 μm  B Combined scores of the mucosal injury in 10-week-old male wildtype and Vil-Ogt KO mice (n=11, ileum: P <0.0001, colon: P = 0.0012)  C Representative images of MPO, F4/80 and CD4 immunohistochemistry in the ileum tissue of male wildtype and Vil-Ogt KO mice. Scale bars = 50 μm  D RT-qPCR of inflammatory markers in jejunum, ileum, and colon (n = 5, jujenum: Il6 P = 0.029, Ifng P = 0.0009; ileum: Tnfa P = 0.0094, Ifng P = 0.0066; colon: Tnfa P = 0.028)  E Immunofluorescent staining of Ki-67 in ileal tissues (n = 5). Scale bars = 50 μm  F, G TUNEL staining (F in the ileum. Quantification of the numbers of positive cells is shown at the right. (TUNEL WT n = 9, KO n = 6, P = 0.0003; Cleaved Cas3 WT = 8, KO = 7, P<0.0001 immunostaining of Cleaved-CASPASE3 (G) in the ileum. Quantification of the numbers of positive cells is shown at the right. (TUNEL WT n = 9, KO n = 6, P = 0.0003; Cleaved Cas3 WT = 8, KO = 7, P<0.0001  H Numbers of survived organoids per 1000 isolated ileal crypts (WT n = 4, KO n = 23, P<0.0001)
Figure 5(A-B) Still images from Movie EV3 of early facial lymphatic development in a lyve1b:EGFP (A), kdrl:nlsmCherry (B) compound transgenic from 42-51.5 hpf, with the CCV-L-derived leading tip cell (green asterisk), the PHS-LP domains (orange and yellow asterisk) and the distal tip of the VA-L (purple asterisk) highlighted (A). Between 42 and 46 hpf, the PHS-LPP (orange outline) cells divide (dark blue parent cells, light blue daughter cells) before sprouting as lymphangioblasts from the PHS and fusing with the tip (pink cells) of the CCV-derived FLS (green outline) at 48 hpf (B). Cells (red) within the VA-L (purple outline) can also be seen migrating towards the now PHS-L-derived FLS tip by 48 hpf. The PHS-LPA is also shown (yellow outline). (C) Quantitation of leading lymphangioblast velocity (µm/min) before and after fusion with another lymphangioblast (CCV-L-derived FLS tip (pink cells) to PHS-L (B, blue cells; n = 5/5) and PHS-L-derived FLS tip (blue cells) to VA-L (F, red cells; n = 5/5)) or by the VA-L to the PHS-L-derived FLS tip (B, n = 5/5). (D) Cell migration tracks where each coloured track depicts the distance (µm) travelled over 3 hours by the leading tip cell in a separate animal before and after fusion with the CCV-L-derived FLS tip (pink cells) and the PHS-Ls (B, blue cells; n = 5/5). (E-F) Still images from Movie EV4 of facial lymphatic development in a lyve1b:EGFP kdrl:nlsmCherry compound transgenic from 48-62 hpf showing that after the CCV-derived FLS fuses to the PHS-L, these take over as the leading tip cells (orange asterisks), with one PHS-L migrating anteriorly to fuse with PHS-LPA (yellow asterisk), and others migrating ventrally to fuse with the VA-L (purple asterisk) After the PHS-derived portion (orange outline) of the FLS (green outline) fuses to the VA-L (purple outline), migration of the entire facial lymphatic pauses to allow for lymphangioblast proliferation (dark blue and red parent cells, light blue and pink daughter cells) before resuming migration anteriorly along the ventral base of the eye (G-H) Cell migration tracks where each coloured track depicts the distance (µm) travelled over 3.5 hours by the leading tip cell in a separate animal before and after fusion with another lymphangioblast (PHS-L-derived FLS tip (blue cells) to VA-L (F, red cells; n = 5/5)) or by the VA-L to the PHS-L-derived FLS tip (F, n = 5/5).