EMBO/sd-character-level-ner huggingface dataset:
PIAS1 Is a SUMO E3 Ligase for HTT.Figure 4(A) Under limiting SUMO conditions (1/4 SUMO-1, lanes 3-9), PIAS1 increases HTT-SUMO modification above 1/4 SUMO alone. Purified HTT (arrow) and SUMO-HTT (boxed region) were detected using anti-HTT. Graph depicts quantitation of the Ni-NTA western blot using the Odyssey Infrared Imaging Software (LI-COR) to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100. (B) Western analysis of overexpression of HTTex1 (46QP-H4 or 3R), SUMO-2 (GFP-SUMO-2), and all the PIAS proteins (PIAS1, PIASxα, PIASxβ, PIAS3, and PIASy). Under nonlimiting SUMO-2 conditions, PIAS1 enhances SUMO modification of HTT. WC TCA shows overall myc-actin (transfection control) and HTT levels. Graph quantitating the ratio of HTT purified versus the HTT modified using the Odyssey (LICOR). (C) Left panel is the autoradiography results of a GST pull-down assay showing that radiolabeled human PIAS1 interacts with HTTex1p. Right panel is a phosphorimager analysis of GST pull-downs, performed in triplicate, showing the percentage of 35S-labeled PIAS protein that bound the GST proteins: GST alone, unexpanded HTT with and without the proline-rich region (20QP and 20Q), expanded HTT with and without the proline-rich region (51QP and 51Q), and the proline-rich region alone (Pro). Error bars were calculated as an estimate of SE = STDEV(n)/SQRT(n-1).
Figure 3(A) Normal proline but decreased whole-brain glycine levels in the brains of PtenΔC/ΔC mice (P21), as determined by ELISA analysis of the S1 fraction, obtained after the removal of unbroken cells and large debris (see Materials and Methods for details). (proline, n = 10 mice for WT and 8 for ΔC, glycine, n = 9 mice for WT and 8 for ΔC, *P < 0.05, ns, not significant, Student's t-test). The error bars represent SEM. (B,C) Decreased extracellular levels of proline and glycine in the brains of PtenΔC/ΔC mice (2-4 month), as determined by microdialysis in the hippocampus (see Materials and Methods for details). (proline, n = 13 mice for WT and 8 for ΔC, **P < 0.01, Student's t-test; glycine, n = 9 for WT and 5 for ∆C, **P < 0.01, Student's t-test, Mann-Whitney U test). The error bars represent SEM. (D) Increased repetitive climbing in juvenile and adult PtenΔC/ΔC mice at P30 (juvenile) and 2-4 months (adult), as indicated by frequency and time spent climbing in Laboras cages, where mouse movements were continuously monitored for 72 hours. Shaded and unshaded periods; 12-hour light-off and light-on periods over 72 hours. (n = 22 mice for WT and 18 mice for ΔC for P30, and 25 for WT and 27 for ΔC for 2-4 months, *P < 0.05, **P < 0.01, ns, not significant, Mann-Whitney U test, Student's t-test). The error bars represent SEM.
Figure 6(a-c) In situ structures of the 96-nm axonemal repeat from the principal piece of pig (a), horse (b), and mouse (c) sperm after classification focused on the ODF attachment. (d-l) Axoneme structures with particles from tomograms from (d,g,j) only the midpiece, (e,h,k) only the proximal principal piece, and (f,i,l) only the distal principal piece. Left panels (d-l) show transverse slices through sperm flagella, while right panels (d'-l') show subtomogram averages of the 96-nm axoneme repeat from the corresponding regions of the tail. In the left panels, one doublet-ODF pair are traced to emphasize the gap between the ODF and the doublet in the midpiece. The ODF is traced in grey with a black outline, and the doublet is traced in blue (light blue for the A-tubule, dark blue for B-tubule). Doublets 3 and 8 are numbered to aid visualization.
Figure 4FLAG-tagged CYLD or catalytically inactive FLAG-CYLD (C598A), respectively, were stably expressed in CYLD-/-MEF, followed by infection with control retrovirus, or retrovirus encoding SPATA2, and purified by FLAG-IP. Purified CYLD or the inactive mutant were added to recombinant di-ubiquitin for 0, 20 or 40 min at 37°C as indicated. After electrophoresis, the gel was cut and the lower part was silver stained to visualize mono-and di-ubiquitin, while the upper part was transferred to a membrane and probed for FLAG and V5. In (A), the experiment was done with M1-linked di-ubiquitin substrate, in (B) K63-linked di-ubiquitin substrate was subjected to the assay, and in (C) K48-linked di-ubiquitin substrate was analyzed.
Transcriptional profiling reveals that Yap regulates the expression of glut1 and glut2.Figure 4Heatmap of RNAseq analysis illustrating statistically significant differential gene expression between WT and yap-/- mutant larvae at 3 dpf. Blue indicates decreased expression, while red demonstrates increased expression Heatmap analysis of RNAseq data highlights the reduction of glucose transporters (glut1 and glut2) in yap-/- mutant larvae at 3 dpf qPCR analysis of glut1 and glut2 expression in WT and yap-/- mutant larvae at 3 dpf. n>3, *p<0.05, ***p<0.001, two-sided Student's t-test; values represent the mean ± s.e.m qPCR analysis of glut1 and glut2 expression in WT, hs:dnYap and hs:Yap transgenic embryos heat shocked at 24 hpf and isolated at 30 hpf. n>3, *p<0.05, **p<0.01, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m qPCR analysis of glut1 and glut2 expression in liver tissue dissected from WT, yap-/- mutant and lf:Yap transgenic adults. n>3, *p<0.05, **p<0.01, two-sided Student's t-test; values represent the mean ± s.e.m
Figure 1(A) Overlap of phosphoproteins that were "up-regulated" by nitrate at 5 min (dark yellow) and 20 min (light yellow) (B) Overlap of phosphoproteins that were "down-regulated" by nitrate at 5 min (blue) and 20 min (light blue). (C) Identification of common phosphoproteins with published phosphoproteome datasets in response to nitrate-deprivation (Menz et al, 2016; Wu et al, 2017) or nitrate-resupply (Engelsberger & Schulze, 2012). In the Sungear figure, vertices represent the different phosphoprotein sets identified in different studies. The circles with arrows within the square represent different intersections among the phosphoprotein studies and the size is proportional to the number of proteins in that circle. The position and the arrows of the circle indicated which vertices or data sets the proteins belong to. The largest circles on the perimeter indicate most of the phosphoprotein are associated with only one experiment.
Figure 1Male, 12wk wildtype (WT; C57Bl/6J) or obese/diabetic monogenic (db/db; BKS.Cg-m+/+ Lepr DB/J; N = 4/group, A-E), New Zealand Black (NZB) and polygenic obese/prediabetic New Zealand Obese (NZO; n=4/group; F-J), as well as young (i.e. 3 mo) and aged (i.e.22 mo; n = 5/group; K-O), mice were fed ad libitum (fed) or fasted for 24h (fasted). Serum non-esterified fatty acids (A, F, K), triglycerides (B, G, L) and ketone bodies (C, H, M) were measured. In addition, serumacylcarinitine profiling was conducted and medium-chain (D, I, N) and long-chain (E, J, O) acylcarnitine concentrations are shown. In another cohort of mice, ex vivo long-chain fatty acid (LCFA) metabolism, including uptake (P), oxidation (Q) and non-oxidative LCFA disposal (NOFAD; R), in precision cut liver slices from fed and fasted WT and db/db mice (n = 3/ group; 4 slices per mouse), were determined. In addition, in slices from fasted mice, glucose output was determined in the presence of incubation with NEFA (BSA-NEFA) or vehicle (BSA) (S).
NexCre cTKOs show agenesis of the corpus callosum and abnormal hippocampal laminationA: Stereological evaluation of cortical volume of young (Y-WT, Y-LM, Y-cTKO, age: 5-6 months, each group n=5) and old (O-LM n=5, O-cTKO n=6, age: 18-20 months) mice in 12 equidistant bins along the caudal-rostral axis of the coronally sectioned brain.Figure 1A: Stereological evaluation of cortical volume of young (Y-WT, Y-LM, Y-cTKO, age: 5-6 months, each group n=5) and old (O-LM n=5, O-cTKO n=6, age: 18-20 months) mice in 12 equidistant bins along the caudal-rostral axis of the coronally sectioned brain. Each bin would comprise an approximately 570 µm thick slab of the brain. No significant differences between genotypes or age groups were seen in the volumes per bin or in the volume distributions along the caudal-rostral axis. Areas shaded in grey correspond to the 95% confidence intervals. B: Double-IHC of coronal brain sections (40 μm) of a littermate (LM) and a NexCre cTKO mouse (age: 5 months) stained for Ctip2 (green, expressed in layer V/VI) and Calbindin (red, expressed in layer II/III and to some extent in layer V). Note that no gross abnormalities in cortical lamination were observed. Layers of DAPI stained cell nuclei are denoted on the right (I-VI), scale bar: 100 µm. C: Giemsa-stained, glycolmethacrylate-embedded coronal brain sections (bregma -1.9) displaying the hippocampus and adjacent callosal fiber tracts of a wildtype (WT, top), littermate control (LM, middle) and a NexCre cTKO (cTKO, bottom; age: 5-6 months). Note the agenesis of the corpus callosum (no CC) in NexCre cTKO mice, which should always be present at this bregma and in sections in which the medial habenula (mHb) is present (CC in WT and LM). While WTs and LMs show a compact layer of CA1 cells, NexCre cTKOs display a bilaminar cytoarchitecture (boxed regions marking higher magnification to the right) with scattered ectopic cells in the stratum radiatum (white arrowheads). In addition, the CA3 appears less compact (red arrowheads). Layer identity visualized via Calbindin staining (right panel, scale bar: 50 µm). D: Quantification of the number of CA1 cells in WT (white, n=5, age: 5-6 months), LM (grey, n=5, age: 18-20 months) and NexCre cTKO (red, n=6, age: 18-20 months) mice. One-way ANOVA revealed no significant difference between genotypes (p = 0.0814). Data are represented as mean + SD. E: Quantification of callosal malformations. n = 10 LM and n = 11 NexCre cTKO animals.
Figure 2pWOX5::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with propidium iodide (PI) staining (red). pSCR::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red). pSCR::GFP-ER expression in Col (WT) and hag1-6 root tips at various time points on CIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red). WOX5 transcript levels in Col and hag1-6 hypocotyl (D) and cotyledon (E) explants at indicated days on CIM. SCR transcript levels in Col and hag1-6 hypocotyl (F) and cotyledon (G) explants at indicated days on CIM.
Analysis of Mtw1 interface mutants in vivo (A) Structure of the Mtw1c head I bound to Mif2 and list of key residues constituting Interface I (Mif2 binding) and Interface II (putative Ame1 binding) from Dimitrova et al.Figure 3Structure of the Mtw1c head I bound to Mif2 and list of key residues constituting Interface I (Mif2 binding) and Interface II (putative Ame1 binding) from Dimitrova et al. Analysis of these Mtw1 mutants in vivo using the anchor-away technique. Mtw1-FRB strains containing RPL13-FKBP12 for cytoplasmic anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 °C. Tetrad analysis of Mtw1-wt or mutants. Surviving spores in which the Mtw1-wt or mutant rescue construct was the only source of Mtw1 are indicated in pink circles. Western blotting confirming the expression of Mtw1-6x-Flag wt or mutant proteins in the Mtw1-FRB strain background. Analytical SEC runs and accompanying SDS-PAGE of Mtw1c-wt or -5A mutant (blue), AO (grey) or a stoichiometric combination of both complexes (green) at 10 µM each. Boxes indicate corresponding fractions in left and right panel. Note that the same Ame1-Okp1 elution profile and SDS-PAGE (middle panel) is displayed in both sets to improve clarity.
Fig.Figure 2(A) A schematic view of the genomic region around Ddc. 5 chromosome deficiencies including Df(2L) ED1203, Df(2L) Exel6042, Df(2L) Exel8040, Df(2L) BSC332 and Df(2L) Exel7071 are shown. A suppressor of Myc depletion-induced male-male courtship was mapped to the region 37C1-37C5, which includes Ddc and 7 other genes. Chaining indices of the indicated genotypes. Statistical significance was compared with relative control. n = 6, 11, 8, 5, 10, 5 (B) Chaining indices of the indicated genotypes. Statistical significance was compared with relative control. ; n = 8, 8, 7 (C) Courtship indices for one target (Canton-S) male and one tester male of the indicated genotypes. Changes were determined by comparing the data with relative control. n = 8, 13, 13 (D) Chaining indices of the indicated genotypes. Statistical significance was compared with relative control. ; n = 15, 23, 15, 15, 6 (E) Courtship indices for one target (Canton-S) male and one tester male of the indicated genotypes. Changes were determined by comparing the data with relative control. n = 16, 16, 18, 11, 24 (F) Chaining indices of the indicated genotypes. Statistical significance was compared with relative control. ; n = 20, 20, 11 (G) Courtship indices for one target (Canton-S) male and one tester male of the indicated genotypes. Changes were determined by comparing the data with relative control. ; n = 20, 20, 17 (H) flies.
Figure 1(A) Intracellular endogenous Aβ42 (green), Ankyrin-G (AnkG; magenta) and GFP (blue) in siBin1- , siCD2AP- and siControl-treated primary cortical neurons (neurons) expressing GFP immunolabelled at 9 DIV with anti-Aβ42 (clone 12F4) and anti-AnkG, analysed by spinning-disk confocal microscopy. The white rectangles indicate the dendrites (Dd) and axons (Ax) magnified below showing Aβ42 in axons and dendrites outlined based on AnkG (magenta) and GFP (blue), respectively. Scale bars, 10 µm.(B) Aβ42 line profiles in dendrites (Dd) of siControl (grey line), siBin1 (blue line) and siCD2AP (red line) neurons shown in (c).(C) Aβ42 line profiles in axons (Ax) of siControl (grey line), siBin1 (blue line) and siCD2AP (red line) neurons shown in (c). (D) Quantification of Aβ42 (12F4) intensity in cell body (CB), dendrite (Dd) and axon (Ax) (n=5, NCB=44-53, NDd=90-120, NAx=60-74; ****PCB<0.0001 siBin1 vs. siControl, ***PCB<0.001 siCD2AP vs. siControl, ****PDd<0.0001 siBin1 vs. siControl, ****PDd<0.0001 siCD2AP vs. siControl, ****PAx<0.0001 siBin1 vs. siControl, t-test, mean ± SEM). (E) Quantification of extracellular endogenous Aβ40, Aβ42 and of Aβ42/Aβ40 ratio by ELISA analysis of conditioned media of 9 DIV siBin1, siCD2AP or siControl neurons (n=6; *PAβ40=0.0270 siBin1 vs. siControl, *PAβ42/40=0.0378 siBin1 vs. siControl, *PAβ42/40=0.0463 siCD2AP vs. siControl, t-test, mean ± SEM). (F) Endogenous APP and APP-CTFs levels by western blot with anti-APP antibody (Y188) of siBin1-, siCD2AP- or siControl-treated neurons at 9 DIV.(G) Quantification of APP and APP-CTFs levels normalized to APP (n=4; *PAPP=0.0355 siBin1 vs. siControl, ***PAPPCTFs/APP<0.001 siBin1 vs. siControl, ****PAPPCTFs/APP<0.0001 siCD2AP vs. siControl, t-test, mean ± SEM).
Figure 6A, B, C and D. Changes in mRNA expression of Ucp1 (A), Pgc1α (B), Pparγ (C) and Trpv2 (D) in iBAT of WT and TRPV2KO mice before (25oC), after 1 day and 3 days cold (4oC) exposure. Mean ± SEM, n = 5 - 8; * P < 0.05; ** P < 0.01 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction. E and F. Western blot results of UCP1 protein in iBAT from WT and TRPV2KO mice before (25oC) and after 3 days cold (4oC) exposure (E). Comparison of UCP1 protein levels before and after cold exposure in iBAT from WT and TRPV2KO mice (F). Mean ± SEM, n = 6; * P < 0.05 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction. G and H. Averages of core body temperatures (G) and activities (H) of WT and TRPV2KO mice with or without cold (4oC) exposure. Data are presented as mean ± SEM, n = 5; * P < 0.05 vs. WT group. Unpaired Student's t-test.
Figure 1A Schematic representation of the UbG76V-GFP chimeric reporter (yellow for ubiquitin and green for GFP) and its associated mRFP internal control (red). The UbG76V-GFP chimera, which contains a mutation in the terminal residue of ubiquitin, cannot be cleaved. The resulting protein is a substrate for poly-ubiquitination (indicated by the circles labeled with "u") and degradation by the proteasome. High levels of reporter poly-ubiquitination and/or proteasome activity result in little or no GFP fluorescence. B,C Expression via the col-19 promoter of UbG76V-GFP in C. elegans hypodermis from a single integrated transgene at (B) L4+24 hours and (C) L4+48 hours. Wild-type animals are shown. Scale bar, 100 microns. D,E Expression of mRFP in the same animals as (B,C) at (D) L4+24 hours and (E) L4+48 hours. Scale bar, 100 microns. F,G Expression via the col-19 promoter of UbG76V-GFP in C. elegans hypodermis from a single integrated transgene at (F) L4+24 hours and (G) L4+48 hours. Mutants for skn-1(zj15) are shown. Scale bar, 100 microns. H,I Expression of mRFP in the same animals as (F,G) at (H) L4+24 hours and (I) L4+48 hours. Scale bar, 100 microns. J Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals from the indicated time point (in hours) after the L4 stage. Animals were exposed to the indicated knockdown RNAi bacterial strains or a strain that only contained the empty RNAi vector since hatching. *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Error bars indicate SEM. K Quantified epoxomicin-sensitive proteasome activity (difference in slope of turnover of fluorescent chymotrypsin substrate between epoxomicin-treated and untreated samples; see Appendix Figure S2) from lysates of animals treated as in (J). *P<0.001, using ANOVA with Dunnett's posthoc comparison to the empty RNAi vector control, with N=3 trials. Error bars indicate SEM. L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals from the indicated time point (in hours) and the indicated genotypes after the L4 stage, as per (J). *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Error bars indicate SEM. M Quantified epoxomicin-sensitive proteasome activity, as per (K). *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=3 trials. Error bars indicate SEM.
RK-33 induces radiosensitization in preclinical mouse models of lung cancer.Figure 6Schematic describing the Twist1/KrasG12D-inducible mouse model. Confirmation of high expression of Twist1 and DDX3 in the lung tumors of the transgenic Twist1/KrasG12D mouse. Scale bar is 100 μm. Treatment schedule for mice receiving hypofractionated radiation (SABR). Stars are intraperitoneal (i.p.) injections with RK-33. Micro-CT images of transgenic Twist1/KrasG12D mice treated as in (C), before treatment and 1 week after treatment. Tumors are indicated by arrows and confirmed by H&amp;amp;E staining of lung sections (lower panel). Scale bar is 250 μm.Quantification of tumor volume measured by micro-CT in Twist1/KrasG12D mice, as shown in (D). Significance was assessed by two-sided, unpaired t-test. Error bars represent SD. An orthotopic lung tumor model was generated using A549 human lung cancer cells and treated as in (C). Figure displays H&amp;amp;E staining of lung sections from radiation-treated (upper panel) and RK-33- and radiation-treated mice (lower panel). Scale bar is 2 mm.Quantification of tumor burden (as tumor surface divided by total lung surface) in orthotopic A549 lung cancer mouse model, as shown in (F). Significance was assessed by two-sided, unpaired t-test. Error bars represent SD. Treatment schedule for mice receiving fractionated radiation in 10 fractions. Stars indicate i.p. injections with RK-33. Downward lightning bolts indicate 3-Gy radiation fractions. Micro-CT images of transgenic Twist1/KrasG12D mice treated as in (H), before treatment and 1 week after treatment.Quantification of tumor volume measured by micro-CT in Twist1/KrasG12D mice, as shown in (I) and expressed as relative tumor size. Significance was assessed by two-sided, unpaired t-test. Error bars represent SEM.
Twenty-one subcellular endocytic phenotypes identified using computational analysis of single cell images (see also , , Table EV1, Table EV2).Figure 1(A) Diagram of the experimental and computational workflow. Yeast mutant arrays harbouring fluorescently-tagged proteins marking specific endocytic compartments were constructed using the synthetic genetic array (SGA) method and imaged using automated high-throughput microscopy. Image and data pre-processing steps included object segmentation and feature extraction, low-quality object clean-up and data standardization. Positive controls and classification training sets were used to train a fully connected 2-hidden-layer neural network (2NN), allowing assignment of phenotypes at the single-cell level and calculation of penetrance. (B) Illustration of endocytosis process and compartment markers. The four endocytic compartment markers used in this study are indicated: Sla1 as a marker of the protein coat component of the endocytic patch (light purple); Sac6 as a marker of the actin component of the endocytic patch (blue); Snf7 as a marker of the late endosome (orange); Vph1 as a marker of the vacuolar membrane (red). The colours chosen for each marker are used throughout this study. FP: fluorescent protein. (C) Example micrographs of yeast cells for each of the 21 subcellular endocytic phenotypes identified in this study. The relevant markers are listed to the left of the micrographs. Scale bar: 5 µm. (D) Pie charts showing the proportion of specific phenotype mutants (SPMs) that have one or more distinct aberrant phenotypes, and non-phenotype-specific mutants for each of the compartments screened. (E) Pie charts showing the proportion of mutant strains that are morphology mutants for one or more markers (left) and specific phenotype mutants (SPMs) that cause one or more aberrant morphological phenotypes (right). The number of mutants in each category is listed within each section. (F) Box plot illustrating the distribution of the fraction of non-wild-type (WT) cells for specific phenotype mutants grouped by the number of phenotypes they cause. ** denotes a significant difference between two groups (p-value < 0.01; significance was determined using analysis of variance (ANOVA) with a post-hoc Bonferroni test). Whiskers extend to the 5th and 95th percentile.
Figure 5A. The IDAP1 or IDAP2 mutation causes silencing of the 35S-LUC reporter gene. The reporter gene was introduced into idap1-1 or idap2-1 by crossing. Seedlings grown in MS plates were imaged after being sprayed with luciferase substrate. B. Real-time PCR analysis of the expression levels of the LUC and NPTII reporter genes in the different genotypes in (A). C. Bisulfite sequencing results showing the methylation levels of the 35S promoter in wild-type and different mutant plants. D. Dysfunction of IDAP1 or IDAP2 causes silencing of the 35S-NPTII but not the RD29A-LUC transgene. Seedlings grown in MS plates were imaged after cold treatment at 4 ºC for 48 h. For kanamycin resistance tests, the seeds were planted on MS medium supplemented with 50 mg/L kanamycin and incubated for 2 weeks before being photographed. The idap1-3 and idap2-2 mutants in C24 background (harboring the RD29A-LUC and 35S-NPTII transgenes) were generated using the CRISPR/Cas9 system. E. Real-time PCR analysis of the expression levels of LUC and NPTII reporter genes in the different genotypes in (D). F. The effects of the ros1-4, idap1 and idap2 mutations on expression of the hypermethylated genes or nearby genes. Gene expression levels in the indicated mutants were determined by real-time PCR. TUB8 was used as an internal control. Standard deviations were calculated from three biological replications, *P < 0.05 (Student's t-test).