EMBO/sd-character-level-ner huggingface dataset:
a, Samples from animals with salivary gland-specific expression of p35 (fkh–Gal4/+; UAS-p35/+), n = 18 (left), drpr null animals (+/w; +/UAS-p35; drpr&Dgr5/drpr&Dgr5), n = 10 (middle), and drpr null animals with salivary gland-specific expression of p35 (fkh–Gal4/w; UAS-p35/+; drpr&Dgr5/drpr&Dgr5), n = 16 (right), analysed by histology for the presence of salivary gland material 24 h after puparium formation.figf2a, Samples from animals with salivary gland-specific expression of p35 (fkh-Gal4/+; UAS-p35/+), n = 18 (left), drpr null animals (+/w; +/UAS-p35; drpr∆5/drpr∆5), n = 10 (middle), and drpr null animals with salivary gland-specific expression of p35 (fkh-Gal4/w; UAS-p35/+; drpr∆5/drpr∆5), n = 16 (right), analysed by histology for the presence of salivary gland material 24 h after puparium formation. Red circles, cell fragments; yellow circle, gland fragments. a, Samples from animals with salivary gland-specific expression of p35 (fkh-Gal4/+; UAS-p35/+), n = 18 (left), drpr null animals (+/w; +/UAS-p35; drpr∆5/drpr∆5), n = 10 (middle), and drpr null animals with salivary gland-specific expression of p35 (fkh-Gal4/w; UAS-p35/+; drpr∆5/drpr∆5), n = 16 (right), analysed by histology for the presence of salivary gland material 24 h after puparium formation. Red circles, cell fragments; yellow circle, gland fragments. b, Quantification of data from a. c, Salivary glands dissected from animals expressing drprIR specifically in GFP-marked clone cells (heat shock (hs) flp/w; UAS-drprIR/+; act,cd2, FRT > Gal4, UAS-GFP/+) 6 h and 14 h after puparium formation. Stained with GFP antibody (green), to label cells expressing drpr IR, and Lamin antibody (red).
Figure 6A) Hierarchy of BAX and DRP1 recruitment to mitochondria in apoptotic cells. Confocal microscopy image of U2OS BAX/BAK DKO cells transfected with mCherry-DRP1 (red), mEGFP-BAX (green) and 4xmts-mTurquoise (cyan) to label mitochondria. After apoptosis induction, translocation of DRP1 to mitochondria and foci formation (set to 0 min) was observed before BAX foci formation (5 min later). Zoomed images correspond to crop regions as indicated. Arrowheads highlight co-localizing foci of DRP1 and BAX. Scale bar 20 µm, zoomed images 2 µm. B) Representative confocal microscopy image of BAX foci formation in apoptotic U2OS BAX/BAK DKO cells transfected with mEGFP-BAX (green) and 4xmts-mTurquoise (cyan) after siRNA-mediated DRP1 depletion. Scale bar 20 µm. C) Forced dimerization of BAX and DRP1 in healthy cells induces their translocation to mitochondria and foci formation. Confocal microscopy images of U2OS BAX/BAK DKO cells transfected with FKBP-mCherry-DRP1 (red) and FRB-EGFP-BAX (green) and stained with MitoTracker Deep Red FM (cyan) were acquired before (0 min) and after induction of BAX/DRP1 dimerization (5-60 min). From top to down, the individual rows correspond to fluorescence signal of BAX and DRP1 and the merge of both together with the MitoTracker signal, respectively. Scale bar 20 µm. D) Quantification of mitochondrial morphology normalized to the number of transfected cells before (Ctrl) and after induced dimerization of BAX and DRP1 (Dim). Significance was tested using paired two-tailed Student´s t-test (** p < 0.01) compared to untreated control. E) Induced dimerization of BAX and DRP1 causes mitochondrial depolarization. U2OS BAX/BAK DKO cells were transfected as described in C) and stained with the mitochondrial membrane potential-sensitive dye MitoSpy NIR (cyan). Confocal microscopy images were acquired before (0 min) and after induced dimerization of BAX and DRP1 (10 min). Scale Bar 20 µm. F) Quantification of mitochondrial depolarization normalized to the number of transfected cells before (Ctrl) and after induced dimerization of BAX and DRP1 (Dim). Significance was tested using paired two-tailed Student´s t-test (* p < 0.05) compared to non-induced control. G G-K) Induced dimerization of DRP1 with itself (G), BAX with itself (H), BAX with TOM20 (I) in U2OS BAX/BAK DKO cells, or BAX with DRP1 in HCT OctaKO cells (J,K), transected with FKBP- and FRB-mCherry DRP1 (G, red), FKBP- and FRB-EGFP-BAX (H, green), TOM20-mCherry-FKBP (red) and FRB-EGFP-BAX (green, I), respectively. Mitochondria were stained using MitoTracker Deep Red FM or MitoSpy NIR (cyan) as indicated. Scale bar 20 µm (G-I) and 10 µm (J,K ). All images are representative of n=3 independent experiments.
Figure 3A) Histogram of the likelihood ratio p-values for the test on differences between WT and mdx at any time point. B) Distribution of the estimated logFCs for mdx versus WT mice at weeks 6, 12, 18, 24 and 30 for the 1532 genes with significant differences between WT and mdx. C) Venn diagram showing the overlap between the sets of genes with significant differences between WT and mdx mice at different weeks. D) Trajectory plots comparing the trajectories of Chordc1 and Psat1 in WT and mdx mice. E) Trajectory plots comparing the trajectories of Cd59a, Chordc1, Hpgd, Ldhb, Lincred1, Myo6, Slc39a11 and Tlr1 in WT, mdx++ and mdx+- mice. F) Scatter plots comparing the estimated logFC of mdx++ vs WT (x axis) and of mdx+- vs WT (y axis) at weeks 6, 12, 18, 24 and 30. Pathway analysis (G) performed by Ingenuity Pathway Analysis of the comparison between mdx and wt mice. H) upstream regulator analysis (H) performed by Ingenuity Pathway Analysis of the comparison between mdx and wt mice.
Confocal microscopy analysis of BMDCs after phage uptake.Figure 4BMDCs were incubated with TRITC-conjugated fdWT (A, C) or fdsc-αDECbacteriophages (B, D) for 6 h. Cells were then fixed, permeabilized, immunostained with anti-EEA-1 (A, B) or anti-LAMP-1 (C, D) (green) antibody and analyzed by confocalmicroscopy. Representative images are shown. Numbers at the bottom of the right panels indicate the percentage of EEA-1 (A, B) or LAMP-1 (C, D) bacteriophage co-localization (mean values ± SD, N = 2, n = 100). Scale bars, 10 mm. White arrows indicate LAMP-1-positive structures that also are positive or contain fdsc-αDEC or fdWT bacteriophages.
Figure 3(A) CCT co-precipitates with A3A but not A3B or A3G. 293T cells were transfected with empty vector (V), A3A-HA, A3B-HA, or A3G-HA. CCT IP was performed from transfected cells and evaluated by immunoblot analysis using antibodies for CCT5 and HA. (B) A3A, but not other APOBEC3 enzymes, interacts with CCT complex subunits. After transfection of 293T cells with HA-tagged APOBEC3 expression plasmids, IP was performed with anti-HA antibody and analyzed by immunoblot using antibodies to HA and CCT subunits. Arrows mark location of each APOBEC3 protein on HA immunoblot. (C) HA immunoprecipitation was performed from U2OS cells with dox-inducible A3A, A3A-C106S (catalytically inactive mutant), and A3B transgenes. IP lysates were evaluated by immunoblot using antibodies to HA and CCT1. (D) Immunoprecipitation of HA-tagged A3B constructs transfected into 293T cells. IP of A3B-HA and A3B-E24R-HA (cytoplasmic localization) was evaluated by immunoblot analysis using antibodies to A3B and CCT1. Arrow indicates CCT5 band. (E) CCT interacts with A3A both with and without interferon. Immunoprecipitation of A3A-HA in K562 cells treated with and without type I interferon (IFN). Anti-ISG15 antibody serves as a control for IFN induction. (F) CCT co-precipitates with endogenous A3A. Immunoprecipitation of the CCT complex in BICR6 cells was analyzed by immunoblot using antibodies to endogenous A3A and CCT1. Chemiluminescent exposure times described as short and long are indicated beside the blot. IgG antibody was used as a control throughout.
Functional interaction between Drosophila Autophagy‐specific gene 1 and Drosophila S6 kinase.Figure 2(A) Quantification of adults that survived after eclosion for denoted genotypes; n=3. Bars indicate mean±s.d. Fifteen larvae of each genotype were examined in each experiment. (B,C) Activation of dS6K in DmATG1 mutants. Phosphorylation of dS6K was examined by immunoblot analyses using phosphospecific dS6K T398 antibody in the third instar larvae or pupae of denoted genotypes. Tubulinimmunoblot was used as a loading control. Rheb expressing flies (hs>Rheb) were used as a positive control (C). hs>Rheb larvae were subjected to heat shock at 37°C for 1 h and then incubated at 30°C for 3 h before sample preparation to induce Rheb expression. For quantification, the levels of dS6K phosphorylation in each genotype were measured using Adobe Photoshop, and normalized to the tubulin levels. Results are expressed as a fold change compared with the wild‐type controls (w1118). DmATG1, Drosophila Autophagy‐specific gene 1; dS6k, Drosophila S6 kinase.
The multifaceted m152 protein selectively modulates STING-dependent type I IFN, but not NF-κB signaling (A) Upon stimulation with DNA or by viral infection, the pattern recognition receptor (PRR) cGAS produces the second messenger 2'3'-cGAMP, which binds to the ER-resident protein STING.Figure 9(A) Upon stimulation with DNA or by viral infection, the pattern recognition receptor (PRR) cGAS produces the second messenger 2'3'-cGAMP, which binds to the ER-resident protein STING. STING then dimerizes and translocates from the ER to the Golgi, from where it activates the signaling pathway leading to induction of type I IFN via the kinase TBK1 and the IRF3 transcription factor. cGAS-STING signaling also induces activation of the NF-κB transcription factor, leading to proinflammatory cytokine expression, but from which subcellular compartment this signaling pathway is initiated is not understood. (B) The viral type I membrane protein m152 is expressed immediately after MCMV infection. m152 binds STING in the ER via their respective luminal domains and traffics with STING to the Golgi. In the presence of m152, trafficking of STING is delayed, leading to a reduced type I IFN, but intact NF-κB response. Mutation of K288 in STING results in a STING mutant that cannot leave the ER and thereby cannot activate the type I IFN response, but is still able to induce the NF-κB pathway. These results suggest that STING induces the NF-κB pathway from the ER, prior to its trafficking to the Golgi.
Figure 3(A) Skin fibroblasts(left) from IV:13, homozygous for the TECRLc.331+1G>A mutation were reprogrammed to hiPSCs(center), which expressed the pluripotency markers NANOG and SSEA4(right). Scale bars: 100 μm. (B) TECRLHom-hiPSCs have a normal karyotype(left) and PluriTest demonstrates a high pluripotency score and low novelty score for all three hiPSC lines (right). (C) TECRLHom-hiPSCs generate derivatives of mesoderm(left), endoderm(center) and ectoderm(right). Scale bars: 25 μm (D) DNA sequencing confirms TECRLc.331+1G>A in the affected family members. Control (left); Heterozygous (Center); Homozygous (right) (E) Schematic of the protocol used for cardiac differentiation of hiPSCs. (F) ACTN2 immunostaining of CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs. Scale bar: 25 μm. (G) Analysis of RT-PCR products by gel electrophoresis. Amplification products of TECRL exons 2-4 from CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs(left) and coding sequence of TECRL from CTRL- and TECRLHom-hiPSC-CMs(right).
Figure 6(A-F) Cells were either untreated (UT) or treated with neocarzinostatin (NCS) in the presence or absence of PIKK inhibitors. Intensities from 3 independent biological replicates were normalized against reference synthetic standards and are depicted as mean ± SEM.
Figure 1A,B Tumor growth curve (left) and summary of end-point tumor masses (right) of 6-8 week-old wildtype (WT) and Peli1-KO (KO) mice inoculated s.c. with B16F10 (A; WT, n=7; KO, n=6) or E.G7 (B; WT, n=5; KO, n=5) tumor cells. C,D Flow cytometry analysis of CD4+ and CD8+ T cells in CD45.2+ TILs and dLN cells from B16F10 tumor-bearing wildtype and Peli1-KO mice, presented as a representative plot (C) and summary graph based on multiple mice (D; WT, n=6; KO, n=6). E,F Flow cytometry analysis of IFNγ-producing CD4+ and CD8+ T cells in TILs, and dLN cells of B16F10 tumor-bearing (day 19) wildtype and Peli1-KO mice, presented as a representative plot (E) and summary graph (F). (WT, n=6; KO, n=6) G,H Flow cytometry analysis of IFNγ+CD8+ T cells in TILs of E.G7 tumor-bearing (day 24) wildtype and Peli1-KO mice, presented as a representative plot (G) and summary graph (H). (WT, n=3; KO, n=3)
Cellular response to the expression of a synthetic pseudoknot construct.Figure 5Change in expression of chromosomal genes in E. coli cells following induction of PK-lacZ expression (1 mM IPTG). Each point represents a transcript. Differentially expressed genes (mRNA count: P < 0.001 and absolute log2 fold-change > 1.37; translation efficiency: P < 0.01) are highlighted in color and by an alternative point shape (transcriptional regulation: purple cross; translational regulation: orange open circle). Venn diagram of genes significantly regulated transcriptionally and translationally after induction of the PK-LacZ construct. Colors match those in panel A. Change in codon occupancy for cells harboring the PK-LacZ construct after induction by IPTG (1 mM) calculated from the Ribo-seq data. Each point corresponds to a codon, which are ordered by amino acid identity and then by abundance in the genome (left most abundant, right least abundant). Dashed horizontal line denotes no change. Outliers are labelled and highlighted in red (Tukey test: 1.5 times the interquartile range below the first quartile or above the third quartile). Translation initiation rates for all E. coli RBSs in cells harboring the LacZ and PK-LacZ constructs in the absence and presence of IPTG (1 mM). Solid line shows the same initiation rate for both conditions. Dotted lines denote linear regressions for the data with no offset. Fractions of mRNA reads and RPFs mapping to each synthetic expression construct (LacZ and PK-LacZ) and E. coli transcripts, which are divided into three major categories: ribosomal, metabolic, and other functions. Data shown for cells cultured in the absence and presence of IPTG (1 mM).
Rev3l-deficient cells show numerous genomic deletionsA.Figure 7A. Experimental design for the whole genome sequencing of Rev3l-deficient cells. Populations of mouse embryonic fibroblasts Rev3l+/+ and Rev3l-/- were isolated from Rev3l+/+ and Rev3lflox/flox littermates and Rev3l was excised using CRE recombinase. The resulting Rev3l-deficient cells and Rev3l+/+ cells were expanded for 90 doublings. Rev3l+/+ and Rev3l−/− subclones were then isolated for deep sequencing. B. Percentage of rearrangements (deletions, duplications and inversions) identified in Rev3l+/+ and Rev3l−/− subclones. C. Size distribution of the deletions detected in Rev3l+/+ and Rev3l−/− subclones. Poisson test. Deletions were counted from four independent clones. D. Distribution of the deletions detected in Rev3l−/− subclones related to the replication timing in deletions per megabase. Difference between late and early regions is statistically significant (p=0.01454, Poisson test with the correction for different length of intervals through the ratio). Data were calculated from two independent clones.
Gain-of-function GltPh variants.Figure 1(A) A GltPh protomer in the OFS and IFS is shown in semi-transparent surface representation with the scaffold and transport domains colored wheat and blue, respectively. HP1 (yellow) and HP2 (red) are emphasized as cartoons. Bound l-Asp and Na+ ions are shown as green sticks and spheres, respectively. Select amino acids mutated in this study are shown as black spheres. (B) Schematic representation of the GltPh transport cycle showing one protomer. Comparatively rapid and slow steps of the cycle are shaded pink and blue, respectively. (C) The topology of a GltPh protomer showing tested mutation sites (circles) with filled circles corresponding to those shown in (A). (D, E) Representative examples of dose-response curves and time courses. Lines through the dose-response curves are fits to the Michaelis-Menten equation; time courses are fitted to linear equations. Shown are means and standard errors over at least three independent repeats. (F) Fold increase (positive values) or decrease (negative values) in the initial uptake rates of the variants relative to WT GltPh. Circles are averages of technical triplicates. Bars are means over independent repeats (as indicated). The dashed line marks a four-fold increase in the initial rate.
Figure 4 A Genetic interactions between irc5-Δ1 and HR mutants. Logarithmically growing cultures of wild type (W303-1a), irc5-Δ1 (IL012), rad51Δ (MC002), irc5-Δ1 rad51Δ (TB046), rad52Δ (MC006) and irc5-Δ1 rad52Δ (TB047) strains were 10-fold serially diluted and plated onto YPD plates with or without MMS  B Disruption of IRC5 is epistatic to rad18Δ and rad5Δ mutations but not to rev3Δ. Logarithmically growing cultures of wild type (W303-1a), irc5-Δ1 (IL012), rad18Δ (MC019), irc5-Δ1 rad18Δ (TB048), rad5Δ (MC018), irc5-Δ1 rad5Δ (TB049), rev3Δ (MC021) and irc5-Δ1 rev3Δ (TB050) strains were 10-fold serially diluted and plated onto YPD plates with or without MMS  C The effect of irc5Δ-1 mutation on PCNA posttranslational modifications. PCNA and its modified forms were detected using a polyclonal anti-PCNA antibody. Asterisks indicate cross-reacting bands  D Lack of Irc5 decreases formation of recombination intermediates during TS. Wild type (W303-1a), irc5-Δ1 (IL012), sgs1Δ (MC012) and irc5-Δ1 sgs1Δ (TB051) cells were synchronized in G1 with alpha-factor and released in media containing 0.03% MMS. The amount of replication intermediates at ARS305 was analyzed by 2D gel. Schematic representation of major 2D gel signals and DNA fragment analyzed are presented. Measurement of DNA content by FACS and X-molecule quantification are displayed (n=2)  E irc5-Δ1 is epistatic to sgs1Δ. Wild type (W303-1a), irc5-Δ1 (IL012), sgs1Δ (MC012) and irc5-Δ1 sgs1Δ (TB051) strains were 10-fold serially diluted and plated onto YPD plates with or without MMS
Figure 3(A) A cluster of 71 neocarzinostatin (NCS)-induced phosphorylations that were enhanced upon continuous inhibition of DNA-PK. Untreated cells are marked UT. This cluster breaks down into two distinct groups according to their ATM dependence 240 min after NCS addition. Clusters were obtained using K-means algorithm implemented in Perseus on Z-scored intensities. Box plots depict 71 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR). (B) Depicted are phosphopeptides that responded to NCS treatment in A-T cells. No significant elevation was observed in these phosphorylations in A-T cells following continuous inhibition of DNA-PK. Box plots depict 71 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 time the interquartile range (IQR). (C) The cluster was enriched for ATM-dependent phosphorylations and the S/TQ phosphorylation motifs. Enrichment was tested using the Fisher exact test implemented in Perseus (D) STRING-Network representation of the proteins in the cluster. Proteins phosphorylated on the S/TQ motif are marked with a yellow border, and the line thickness represents the combined score for interaction confidence according to STRING. (E) Western blotting analysis of selected phosphorylations in the above cluster showing DNA-PKi-induced elevation. (F) Temporal dynamics of DNA-PK-dependent attenuation of pS824/KAP-1.
MUPs bind ubiquilins.Fig 1(A) Domain structure of human UBQLN1 (Interpro: http://www.ebi.ac.uk/interpro/). (B) Gal4 DNA-binding domain fusions of MUPs were tested with Gal4 activation domain fusions of ubiquilins (murine) in Y2H. (C) HEK293 cells transfected with indicated MUP-V5, myc-UBQLN1 (human), or empty vector were immunoprecipitated (IP) with anti-myc or anti-UBQLN1 antibodies. Western blot (WB) of IP and lysate were probed as indicated. (D-E) WB of input lysate and IP from HEK293 cells expressing myc-UBQLN1 and Rv1926-V5 (D) or Rv1566-V5 (E). IP was performed using antibodies directed against myc, V5, or isotype control (IgG).
Figure 4A. RPE or MCF10A cells were transfected with the indicated combinations of siRNAs and re-seeded to low density, and cell extracts were analyzed by Western blotting for the indicated proteins. B. The cells described in (A) were analyzed by RT-qPCR for the indicated target genes (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test). C. Sparsely cultured RPE cells transfected with the indicated siRNAs were immunoprecipitated with an anti-AMOTL2 antibody and analyzed by Western blotting for the indicated proteins. WCL, whole cell lysates.
Requirement of Age2 ArfGAP activity in AP-3 trafficking.Figure 7(A, B) AP-3 sorting defects in strains overexpressing Gcs1 (A) or Age2 (B). The AP-3 reporter GNS was localized relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm. (C) Localization of AP-3 in ArfGAP overexpression strains. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm. (D) Quantification of Apl5 dots in (C). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean. (E, F) AP-3 sorting defects in GAP-dead mutants of Gcs1 (E) and Age2 (F). The AP-3 reporter GNS was localized relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm. (G) Localization of AP-3 in GAP-dead mutants of Gcs1 and Age2. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm. (H) Quantification of Apl5 dots in (G). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean.
The BDNF mRNA is a miR-16-5p target and is regulated during BDNF stimulationDiagram showing the region of the BDNF 3'UTR bound by miR16 family of miRNAs that is conserved across species.Figure 6Diagram showing the region of the BDNF 3'UTR bound by miR16 family of miRNAs that is conserved across species. Nucleotides colored in red depict the binding site of miR-16-5p (sequences obtained from www.targetscan.org). B) MiR-16-5p targets the 3'UTR of the BDNFmRNA. Cortical neurons were transfected with 100ng pmiRGlo dual-luciferase reporter consisting of the 3'UTR of BDNF (3'UTR WT) or a miR-16-5p-binding site mutant (3'UTR Mut) and either miR-Ctrl or miR-16. The Firefly to Renilla luciferase ratio was measured and values were normalized to the respective reporter expression without miRNA mimic co-transfection (n=3, p=0.02, t-test type 2). C) BDNFstimulation leads to an increase in the translation of 3'UTR WT (p=0.02, t-test type 3) but not 3'UTR Mut luciferase reporter, which is rescued by miR-16-5p overexpression (p=0.002; t-test type 2). Cortical neurons were transfected as in (B), stimulated with BDNF or vehicle control for 20 minutes, and lysed two hours after stimulation (n=4). D) Pre-miR16 is present in the neuronal cell soma and processes of hippocampal neurons. Neurons were plated on compartmentalized chambers and RNA was isolated from the neuronal cell body and processes separately. Pre-miR137 and pre-miR341 were used to assay somatic and neurite enrichment, respectively. Raw Ct values were normalized to GAPDH mRNA in each experiment (n=3).