EMBO/sd-character-level-ner huggingface dataset:
Figure 3The c.2272-18C>A variant generates a new splice acceptor sequence 16 base pairs upstream of the normal exon 23 border, resulting in a frame-shift and premature stop codon. Levels of VPS16 transcripts in fibroblasts by qPCR. The probe of the exon 22-24 PCR spans the misspliced intron-exon border. Data was normalized to levels of POLR2A using the ∆∆Ct method. Quantifications of VPS16 in fibroblast lysates by immunoblotting. Representative immunoblots (bottom) and summary quantifications (top) of the levels of VPS16 in patient A (n=4) and patient B (n=3), normalized to levels of actin (ACTB) and expressed as % of controls. Schematic illustration of HOPS and CORVET complexes showing their subunit compositions. The Rab5-binding CORVET subunits VPS3 and VPS8 replace the Rab7-binding VPS39 and VPS41 of HOPS. Models adapted from Bröcker et al (2012). Quantifications of HOPS/CORVET subunits in fibroblast lysates (top) and representative immunoblots (bottom), analyzed as in (C). (E) VPS33A, (F) VPS11, (G) VPS18. Analysis of VPS33A and VPS11 mRNA levels in fibroblasts by qPCR. Data was normalized to levels of POLR2A using the ∆∆Ct method. Immunoblots (left) and quantifications normalized to control (right) of VPS16, VPS33A and VPS11 in lysates from fibroblasts transduced with a lentivirus to express VPS16 or control (empty vector).
Interaction between endogenous and spatially restricted Bcl‐2 and Beclin‐1 in starvation and after addition of ABT737.Figure 6(A) Immunoprecipitation of endogenous Bcl‐2 and Beclin‐1. Untransfected HeLa or MV4.11 cells were treated by culture in nutrient‐free EBSS or with ABT737 (1 μM). (B) Immunoprecipitation of wild‐type and ER‐targeted Bcl‐2 with Beclin‐1. Cells stably transfected with wild type, ER‐ and mitochondrion‐targeted Bcl‐2 were subjected to starvation or treated with ABT737, followed by immunoprecipitation of Bcl‐2 and immunodetection of Beclin‐1. (C, D) Immunoprecipitation of Bcl‐2 and Beclin‐1 in subcellular fractions. Cells treated as in (B) were lysed and subjected to subcellular fractionation to enrich either ER vesicles (microsomes, (C)) or mitochondria (heavy membrane fraction, (D)), followed by immunoprecipitation of Bcl‐2 and immunodetection of Beclin‐1. Calreticulin and Hsp60 were used as internal controls. Results are representative of three independent determinations.
Figure 2PBMCs from P. falciparum symptomatic (n=16) and asymptomatic (n=24) infected individuals as well as healthy immune controls (n=24) were stained with a panel of metal-labelled antibodies and analysed by CyTOF. tSNE analysis was performed and FlowSOM clustering was used to identify individual cell sub-populations within gated: A. TH1-like memory CD4+ T cells (CD19-CD3+CD4+CD45RA-CCR6-CXCR3+) B. TH2-like memory CD4+ T cells (CD19-CD3+CD4+CD45RA-CCR6-CXCR3-) C. Circulating memory TFH cells (CD19-CD3+CD4+CD45RA-CXCR5+) D. Classical MBCs (CD3-CD19+CD20+CD10-CD27+CD21+) E. Atypical MBCs (CD3-CD19+CD20+CD10-CD27-CD21-) F. Activated MBCs (CD3-CD19+CD20+CD10-CD27+CD21-) The tSNE plots in the top panel display cell density and represent the pooled data for each group, while the lower panel shows a projection of the FlowSOM clusters on a tSNE plot. Heatmaps show the median marker expression for each FlowSOM cluster.
Figure 1(A) Human osteosarcoma U2OS GFP-LC3 cells were treated with a library of polyphenols and polyamines for 6 h And then GFP-LC3 dots were counted to measure autophagy activity. Top 20 hits were shown in the right frame. Human osteosarcoma U2OS cells (B or neuroglioma H4 cells (D stably expressing GFP-LC3 were treated with a library of chalcones (30 µM) as indicated , or were left untreated for 6 h. The cells were then fixed and GFP-LC3 dots were counted as an indicator for autophagy. Data are means ± SD (* = p < 0.05; ** = p < 0.01;*** = p < 0.001). Representative images are shown in (B,D). Scale bar equals 10 µm. (C,E) Acetylation: U2OS and H4 cells were treated as described above, followed by incubation with specific antibodies to block acetylated tubulin; Thereafter immunofluorescence was conducted with antibodies against acetylated lysine residues and appropriate AlexaFluor conjugated secondary antibodies before the assessment of cytoplasmic fluorescence intensities. Representative images of acetylation are shown in (C,E). Scale bar equals 10 µm. Human osteosarcoma U2OS cells F) or neuroglioma H4 cells H) stably expressing GFP-LC3 were treated with a library of chalcones (30 µM) as indicated or rapamycin (10 µM), or were left untreated for 6 h. The cells were then fixed and GFP-LC3 dots were counted as an indicator for autophagy. Data are means ± SD (* = p < 0.05; ** = p < 0.01;*** = p < 0.001). Viability: U2OS and H4 cells were treated with 30 µM of the indicated chalcones for 24 h and then nuclei were stained with Hoechst 33342 and the number of cells harboring normal nuclei (i.e. non pyknotic, "healthy cells") was determined. All chalcones were hierarchically clustered upon z-scoring the following phenotypes: autophagy (number of GFP-LC3 dots), viability (number of healthy cells) and acetylation (cytoplasmic fluorescence intensity of acetylated lysine residues) in U2OS and H4 cells. Results are reported as a heatmap (G, I). (J, K) U2OS cells were treated with 3,4-DC in the presence or absence of dichloroacetate (DCA), α-ketoisocaproic acid (KIC), or L-leucine (Leu). GFP-LC3 dots were quantified in (J). Acetylation staining was performed as previously described, and acetylation intensity in the cytoplasm was measured in (K). Data are means ± SD (*** = p < 0.001 versus DMSO/Ctr; ##=p<0.01, ###=p<0.001 versus 3,4-DC/Ctr).
Figure 5A. Expression from the gadB and recA promoters, measured at the population level using GFP reporters over time; 0.5 μg/mL TMP was added at time zero. The acid stress response (gadB) clearly precedes the SOS response (recA). Error bars show standard deviation of three replicate experiments measured on different days. B. Normalized expression of gadB and recA expression over time in individual cells responding to the addition of 0.5 μg/mL TMP at time zero shown for one microcolony. Dashed line: threshold used to determine response times (Methods). Lower panels, upper row: Dual-color images of gadB (blue) and recA (red) in single cells at 4 different time points. Most cells are predominantly either red or blue, i.e. acid stress and SOS response are typically not both strongly expressed in the same cells. Lower row: Constitutively expressed mCherry used for segmentation. C. Scatter plot of response times for gadB and recA in single cells; there is no clear temporal order and no significant correlation (r = 0.08 ± 0.08, p = 0.68); data is combined from three microcolonies. Error is from subsampling of descendants of single cells present at the time point of TMP addition D. Expression of gadB and recA in inosine-supplemented medium, averaged over 13 single cells in a microcolony. Insets: Dual color image of gadB and recA and segmentation image at t = 6h.
Figure 5A. Structural views suggesting that Mdm12 might have a preference for certain phospholipids. The surface representation of Mdm12 positioned around the binding site for the head group of PE is shown with charge distribution (left) and the sequence conservation (right) in the same orientation. Surface electrostatics and sequence conservation were calculated using an APBS program [31] with the nonlinear Poisson-Boltzmann equation and contoured at ±3kT/e, and ConSurf website (consurf.tau.ac.il) [32] with 34 different yeast orthologs, respectively. The ribbon diagram shown in the middle indicates the overall orientation of Mdm12. B. Lipid displacement experiments to identity the natural ligands of Mdm12in vitro (see material and methods section for details). NBD-PE-preloaded Mdm12-GST was mixed with a series of glycerophospholipids at different concentrations, and displacement of NBD-PE by non-labeled ligand was estimated from the decrease in fluorescence. Means ± SD are shown (n=3 independently performed experiments).
PglZ phylogeny and classification of BREX subtypesShown is a phylogenetic tree of the PglZ protein instances detected in this study.Figure 5A Shown is a phylogenetic tree of the PglZ protein instances detected in this study. The tree is color‐coded according to the operon organization of the neighboring genes (BREX types). The gene order and genomic organization of each type is illustrated next to its relevant branch on the PglZ tree. Numbers depict bootstrap values.B Prevalence of the different BREX subtypes within the BREX superfamily of defense systems among the sequenced genomes that were analyzed.
Figure 1A-E Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread spermatocytes at (A) zygotene (Zyg.), (B) pachytene (Pac.), (C) diplotene (Dip.), (D) late diplotene (L. dip.), and (E) prometaphase I (ProM I). F-H Double-immunolabeling of Sororin (green) and SYCP1 (pseudocoloured in pink) on spread spermatocytes at (F) zygotene, (G) pachytene, and (H) diplotene. I-K Double-immunolabeling of Sororin (green) and HORMAD1 (pseudocoloured in blue) on spread spermatocytes at (I) zygotene, (J) pachytene, and (K) diplotene. The sex bivalent (XY) and their pseudoautosomal regions (arrows), as well as the round body (arrowheads) are indicated. Scale bar: 10 µm.
PYCR1 abundance level is associated with breast cancer relapse.Figure 4A) Abundance level of the Eigengene vector of yellowgreen module. Each bar represents a patient and is separated and colored according to relapse status B) Scatter plot of module membership vs gene significance score for the yellowgreen module that shows highest correlation to relapse. Proline biosynthesis proteins PYCR1 and ALDH18A1 are highlighted. Significance of Pearson correlation was determined using t-test. C) Diagram of the proline metabolism pathways. Proline biosynthesis genes are PYCRL, Pyrroline-5-Carboxylate Reductase Like; PYCR1, Pyrroline-5-Carboxylate Reductase 1; PYCR2, Pyrroline-5-Carboxylate Reductase 2; ALDH18A1, Aldehyde Dehydrogenase 18 Family Member A1 (Pyrroline-5-Carboxylate Synthetase). D) Proline biosynthesis protein levels in breast cancer subtypes (Log2 ratios of light vs. SILAC standard). The upper and lower limits of the box show values from the first to the third quartile, with the horizontal line indicating the median. The whiskers extend 1.5 times the interquartile range from the edges of the box. Outlier values were plotted outside the whiskers. Significance was determined using paired Student's t-test and p values were corrected using permutation based FDR . *p < 0.05 and *** p < 0.001. E) High abundance of PYCR1 in the cancer tissue was validated by immunohistochemistry on matched tissue specimens from patients in this study (n=3); Scale bar: 200 μm. F) Quantification of IHC shows percentage of cells with 2+ and 3+ PYCR1 staining intensity. Data is expressed as mean ± SD of three patient samples. Paired samples were compared by a Student's t-test. *p < 0.05 and *** p < 0.001. G) Kaplan Meier survival curve for PYCR1 levels in residual cancer in the current proteomics data. Cox univariate p value, risk table and hazard ratio with 95% CI indicated.
1,25‐dihydroxyvitamin D3 and retinoic acid induce p19ink4d expression.Figure 33 and retinoic acid induce p19ink4d expression. Expression of p19ink4d messenger RNA by reverse transcription-PCR (A) or protein by western blotting (B) was carried out on extracts of cells treated with 3 (1,25D3) or retinoic acid (RA), as indicated. (C) Treatment with 1,25D3 or RA transiently enhances co‐immunoprecipitation (IP) of p19INK4D with cyclin‐dependent kinase CDK4 in U937 cells. Similar results were obtained in SCC25 cells (not shown). A control western blot (W) of CDK4 levels in ligand‐treated cells is shown below. (D) Treatment with 1,25D3 enhances co‐immunoprecipitation of p27KIP1 with CDK4 in U937 cells. Extracts of U937 cells were probed for p27KIP1 and CDK4, and immunoprecipitated with an anti‐CDK antibody and probed for p27KIP1 co‐immunoprecipitation.
a, Proteomics work flow.f1a, Proteomics work flow. b, diGly sites identified and quantified across 73 experiments. FDR, false discovery rate. c, Log2(H:L) plots for quantified diGly peptides for HCT116PARKIN (experiment 17) or HeLaPARKIN (experiment 57) cells (Supplementary Table 2). d, Overlap of ubiquitylation sites in HeLaPARKIN biological triplicates (1 h CCCP + Btz) (Supplementary Tables 1, 2). Ex, experiment. e, Ubiquitylation site and protein overlap between all HCT116PARKIN and HeLaPARKIN experiments treated with CCCP and Btz for 1 h. f, log2(H:L) ratios for selected diGly sites from HCT116PARKIN (Ex17) and HeLaPARKIN (Ex57) (1 h CCCP + Btz).
miR-23~27~24 clusters are required for ESC differentiation.Figure 7Real-time PCR analysis of Oct4 and several lineage differentiation markers expression during EB formation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3). Beating EB incidence during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean + s.d. (n=3). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001. Real-time PCR analysis of several cardiac markers expression during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3). Actinin, ANP and Troponin Iimmunofluorescence staining of differentiation cultures derived from wild-type and knockout ESCs. Volumes of teratomas at different days after injection. Data are shown as mean + s.d. (n=4). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001. Weight of teratomas derived from wild-type and knockout ESCs. Data are shown as mean ± s.d. ** indicates P-value < 0.01. Oct4 immunohistochemistry staining of teratomas derived from wild-type and knockout ESCs. Representative Oct4 positive regions are shown. Hematoxylin and eosin staining of teratomas derived from wild-type and knockout ESCs. Ectoderm, Mesoderm and Endoderm are marked with different color arrows.
a) Schematic representation of 60Q-HSC70bm.figf1(a) Schematic representation of 60Q-HSC70bm. (b) PC12 cells transfected with either 60Q or 60Q-HSC70bm. The presence of two HSC70bm motifs in 60Q-HSC70bm reduced the number of inclusions as compared to 60Q (scale bar, 20 μm). (c) Quantification of the PC12 cells with inclusions 24 h after transfection with either 60Q or 60Q-HSC70bm. 10 mM 3-methyladenine (3-MA), 10 μM pepstatin A and 10 μM leupeptin were added to inhibit macroautophagy, the proteases, cathepsins D and E, and cathepsin A, respectively. Bars in c represent relative mean values ± s.e.m. from four independent experiments, with levels of aggregation observed for 60Q normalized to a value of 1. Quantifications were performed by ArrayScan.
The synergy between mycolactone and BZ cytotoxicity extends to mouse B cell lymphomas.Figure 5A Effect of mycolactone on v-abl pro-B cell viability after 24 and 48 h of mycolactone treatment, as measured by exposure of Annexin V and incorporation of PI. Data are Mean % of live, apoptotic and dead cells from technical duplicates , relative to total cells. Data information: Data shown are representative of experiments performed with two independent v-abl cell clones, with similar results. B v-abl pro-B cells were treated for 24 h with mycolactone and/or BZ at the indicated concentrations. The percentage of live, apoptotic and dead cells was then measured by flow cytometry Data are Mean % from technical duplicates, relative to total cells. Data information: Data shown are representative of experiments performed with two independent v-abl cell clones, with similar results. C v-abl pro-B cells were treated with mycolactone and/or BZ at the indicated concentrations for 6 h. CHOP mRNA levels were quantified by qPCR. Data are Mean RNA fold changes (2-∆∆CT) ± SD relative to untreated controls. N = 6 (cumulative data of 3 independent experiments with technical duplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Data information: Thapsigargin (Tg, 2 µM, 6h) was used as a positive control. D v-abl pro-B cells were treated for 6 h with mycolactone and/or BZ at the indicated concentrations. ATF4 protein levels were assessed in cell lysates by Western blot (top panel) and quantified relative to GADPH levels (lower panel). Data information: Data shown are representative of experiments performed with two independent v-abl cell clones, with similar results. Thapsigargin (Tg, 2 µM, 6h) was used as a positive control.
Nuclear flattening is sufficient to activate c-Jun and YAP independently of actomyosin contractility and cell spreading area.Figure 3Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. (b) Corresponding quantifications of p-Jun Ser63 nuclear intensity. Representative cells stained for YAP (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. Corresponding quantifications of YAP activity (ratio nucleus/cytosol). Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Blebb, Blebb + AP condition and in Ctrl, Y27632 and Y27632 + AP. Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Cytochalasin D (CytoD), CytoD + AP condition. Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in cells depleted or not for SUN1 and SUN2. Representative cells cultured on circular micropatterns with surfaces of 1500 and 500 µm2 and stained for p-Jun Ser63 (magenta), YAP (cyan) and for DNA (green). Additionally, cells were cultured on the micropatterns of 500 µm2 and an agarose pad was used to flatten their nuclei. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. Corresponding quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity.
Ltn1 is rapidly degraded in a RING domain– and proteasome-dependent manner.Figure 4.(A) Wild-type cells expressing Bre5-GFP, Ltn1-Flag, or ltn1-ΔRing-Flag were starved for the indicated periods of time. (B) Cells were treated for 30 min with 100 µM cycloheximide or 100 µM MG132. Expression of tagged proteins was analyzed by anti-GFP or anti-Flag blotting of whole cell extracts. Five times less extracts were analyzed for ltn1-ΔRing-Flag cells. A nonspecific band served as a loading control. White lanes indicate that intervening lanes have been spliced out. WT, wild type.
Figure 3Two-fold serial dilutions of the total protein extracts from the wild-type, ash122/ash19011 (ash1-), ash122,NSDds46/ash19011,NSDds46 (ash1-, NSD-) and Set21 (Set2-) larval brains, imaginal discs and salivary glands were analyzed by western blot with antibodies against H3K36me1 (A), H3K36me2 (B) and H3K36me3 (C). Note the strong (>10-fold) reduction of H3K36me3 signal in the Set2- extract and the slight (~2-fold) reduction of H3K36me1 signal in the ash1- and ash1-, NSD- extracts. The protein extracts from the wild-type, double ash1-, NSD- and single NSD- and Set2- mutants (right panels) were analyzed together on the same membrane, however the images of the H3K36me1 and H3K36me3 western blots were modified to splice out the marker lane between the ash1-,NSD- and the Set2- extracts. Western blots with constitutively expressed BEAF-32 protein were used as loading controls.
Ccno is dynamically expressed from early phases of ciliogenesis and Ccno deficiency impacts on the transcriptional programme for ciliogenesis A–H X‐Gal‐staining of (A–C) control and (D–H) CcnoTA/+ embryonic lungs at indicated stages showing onset of Ccno expression from E13 in the proximal trachea and in the main bronchi.Figure 3A-H X‐Gal‐staining of (A-C) control and (D-H) CcnoTA/+ embryonic lungs at indicated stages showing onset of Ccno expression from E13 in the proximal trachea and in the main bronchi. Expression is extending to more distal regions, and from E16 staining is also found in bronchioli.I X‐Gal‐staining indicating LacZ expression from the CcnoTA/+ allele in mTEC cultures after 5 days of differentiation‐onset by switching to air-liquid interface (ALI) conditions (ALI d5). J Transcript levels of Ccno and indicated genes with known functions for the generation of multiple cilia during mTEC differentiation. mRNA levels from three independent experiments were measured by qRT-PCR and levels of expression set as 1 on day 0 of ALI cultures. Scales for Ccno and Deup1 are indicated on the left, and for Cep152, Cep63 and Ift57 on the right side. K Relative mRNA expression levels for indicated genes were measured by qRT-PCR in wild‐type and Ccno‐deficient mTEC cultures at indicated days after differentiation‐onset. Relative values were calculated as in (J) relative to day 0 of ALI cultures.
TEs frequently insert in genes and regions of open enhancer-like chromatin A The distribution of somatic TE insertion sites on the Drosophila chromosomes.Figure 6A The distribution of somatic TE insertion sites on the Drosophila chromosomes. Dark grey boxes represent heterochromatic regions. B Somatic TE insertion sites of rover LTR elements were depleted from intergenic and exonic sequences and enriched in introns and 3'UTR regions of the fly genome. Insertions of copia LTR elements were also enriched in introns. C Selected genes relevant for the gut physiology with putative somatic insertion sites. Genic regions +/- 500bp were considered. D Correlations of somatic insertion sites of the three most represented TE families (rover, copia and I-element) with modENCODE tracks for adult fly tissues. E Correlations of somatic insertion sites of the three most represented TE families (rover, copia and I-element) with DamID tracks for adult fly intestinal stem cells (ISC).
a,b) Overexpression of Fbxo7 by da-GAL4 (da>Fbxo7) suppresses climbing (a) and flight (b) defects of parkin mutants.figf3(a,b) Overexpression of Fbxo7 by da-GAL4 (da>Fbxo7) suppresses climbing (a) and flight (b) defects of parkin mutants. (c) Overexpression of Fbxo7 also suppresses dopaminergic (DA) neurodegeneration parkin mutants. (d-f) Top and middle panels, toluidine blue-stained sections of adult thorax. Bottom panels, transmission electron micrographs of muscle. Fbxo7 overexpression suppresses muscle degeneration and mitochondrial disruption in parkin mutants. Scale bars: 200 μm (top), 20 μm (middle), 2 μm (bottom). Images are representative of three animals per genotype. (g,h) Overexpression of Fbxo7 pathogenic mutants, mt-MTS or isoform 2 by da-GAL4 fails to rescue climbing (g) and flight (h) deficits in parkin mutants. Control genotype is park25/+; da-GAL4/+. All transgenic lines are site-directed integration except WT(4), which is random integration line 4 (see Supplementary Fig. 3 and Online Methods). Histograms indicate mean ± s.e.m. One-way ANOVA with Bonferroni correction (***P 0.001, **P 0.01). For climbing and flight assays, at least 50 flies were assessed; number of flies is shown for each bar.