EMBO/sd-character-level-ner huggingface dataset:
Figure 7(A) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. P-TBK1, magenta; NBR1, green; Hoechst, blue. (B) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue. (C) Representative confocal micrographs (as maximum intensity projections) from wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1 and transduced with indicated sgRNA. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue. (D) Quantitation of colocalization between NBR1 with FIP200 in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C) Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). sgATG9A samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. n > 200 NBR1 puncta for each biological replicate. (E) Quantitation of FIP200 intensity at NBR1 puncta in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C). Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). n > 80 FIP200-positive puncta for each biological replicate. Samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. a.u., arbitrary intensity units. (F) TAX1BP1 drives local autophagosome formation in the context of NBR1 puncta. This activity is independent of LC3 or the ubiquitin-binding domains of TAX1BP1. Rather, a newly defined NBR-interacting domain (or N domain, not shown) drives the association of TAX1BP1 with NBR1 puncta. Subsequent recruitment and clustering of FIP200 by TAX1BP1 induces local autophagosome formation and enforces cargo specificity, thereby replacing the requirement for lipidated LC3.
Netrin-3 is a target gene of MYCN in NB.Figure 3A. Gene Set Enrichment Analysis (GSEA) in high netrin-3 NB patients. The cohort was dichotomized with the n=50 lowest vs 50 highest: amplification the MYCN pathway (p=0.039; fdqr=0.025). C. Q-RT-PCR analysis of netrin-3 gene expression in IMR32 (n=5) and IGRN91 (n=5) NB cell lines after MYCN silencing by siRNA (U-test). Error bars indicate s.e.m. D. ChIP-seq analysis of MYCN binding, active enhancer epigenetic marks H3K27ac, H3K4me1, and active promoter epigenetic mark H3K4me3, on netrin-3 locus (red line). An enrichment of MYCN, associated with active enhancer marks, was detected in three different neuroblastoma cell lines NB-1643, COGN415 and LAN5. E. Quantitative analysis showing the size of IGRN91 primary tumors implanted on CAM and silenced or not for netrin-3 (n=11 siScr, n=9 siNetrin-3; U-test). Error bars indicate s.e.m. F. Quantitative analysis showing the size of IMR32 primary tumors silenced or not for netrin-3 (n=5 siScr, n=7 siNetrin-3; U-test). Error bars indicate s.e.m.
Expression of R3hdml is controlled by the expression of MyoD.Figure 2A Cultured C2C12 cells were transfected with the firefly luciferase construct containing the 781 bp fragment upstream of the translational start codon of the R3hdml coding sequence (R3hdml-luc), together with Renilla luciferase vectors as controls; the cells were then differentiated in the presence of 2% horse serum. Cells were harvested at the indicated time points, after which luciferase activity of the cell lysate was measured. B The table represents the schematic of each construct. All inserts in the constructs contain the same 3ʹ end. The 781-bp fragment of the 5ʹ flanking sequence from the murine genome, which contains several putative MyoD binding sites (black box), was subcloned into a luciferase reporter vector (Construct C). We also subcloned the genomic sequence containing one high affinity MyoD binding site CACCTG (Construct B) and one without a high affinity MyoD binding site (Construct A) into a luciferase reporter vector. C Plasmids containing MyoD cDNA or empty vector were transfected into C2C12 cells. One day and 3 days after transfection, cells were lysed and subjected to SDS-PAGE and western blotting using anti-MyoD antibodies. D Plasmids containing MyoD cDNA or empty vector together with R3hdml-luc and Renilla luciferase vectors were transfected into C2C12 cells. One day and 3 days after transfection, the luciferase activity of the cell lysate with or without MyoD overexpression was measured. Relative luciferase activity is expressed as the fold activity above the background conferred by the empty vector together with R3hdml-luc.
The lifespan-extending and autophagy-inducing effects of spermidine in yeast are not mediated by Sir2.Figure 2.The lifespan-extending and autophagy-inducing effects of spermidine in yeast are not mediated by Sir2. (A-E) EGFP-Atg8p was ectopically expressed in wild-type (WT) or Δsir2 S. cerevisiae undergoing chronological aging on small synthetic 2% glucose media with or without (Co, control) supplementation of 4-mM spermidine (Spd). (A) Representative images. EGFP-Atg8p localization (bottom) was visualized by fluorescence microscopy. Yeast cells undergoing autophagy (in which EGFP-Atg8p exhibits a prominent vacuolar localization) are indicated by arrows. Yeast morphology was monitored by differential interference contrast (DIC; top). (B) Representative immunoblots against EGFP. Free EGFP indicates the vacuolar degradation of EGFP-Atg8p fusion, thereby representing the autophagic flux. Notice that both WT and Δsir2 yeast cells show similar free EGFP levels after spermidine-mediated autophagy induction. (C) Relative alkaline phosphatase (ALP) activity indicative of autophagy. n = 3. (D) Survival data. n = 4. (E) Quantification of reactive oxygen species. Bars indicate the percentages of cells exhibiting the reactive oxygen species-mediated conversion of dihydroethidine (DHE) into ethidium (Eth; n = 4). Data represent means ± SEM; *, P < 0.001 as compared with untreated cells of the same genotype. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. RFU, relative fluorescence unit.
Figure 3 A. ChIP-Seq analysis of Rif1 and Rif1-∆C594 proteins shows tRNA gene and origin binding, with widened peaks at early origins ARS606 and ARS607 in HU block. Plots show Chromosome VI genome coordinates 160,000- 210,000. Plot colours here and in following Figures are as in Fig 2B. Widened peaks are not observed in unperturbed S phase samples, or for Rif1-∆C594   B. Association of Rif1-∆C594 at replication origins is enhanced in HU block. ChIP enrichment of Rif1 and Rif1-∆C594 around early origin ARS1426 (left) and late origin ARS1412 (right)   C. Differential association of Rif1 and Rif1-∆C594 to centromeres. ChIP enrichment of Rif1 and Rif1-∆C594 around the CEN4 locus
2019-nCoV-S utilizes ACE2 as cellular receptor.Fig. 2.(A) The S protein of 2019-nCoV clusters phylogenetically with S proteins of known bat-associated betacoronaviruses (see also SI Figure 1 for more details). (B) Alignment of the receptor binding motif of SARS-S with corresponding sequences of bat-associated betacoronavirus S proteins that are able or unable to use ACE2 as cellular receptor reveals that 2019-nCoV possesses amino acid residues crucial for ACE2 binding. (C) 293T cells transiently expressing ACE2 of human (dark blue) or bat (light blue) origin, human APN (purple) or hDPP4 (green) were inoculated with pseudotyped VSV harboring VSV-G, SARS-S, 2019-nCoV-S, MERS-S or 229E-S. At 16 h postinoculation, pseudotype entry was analyzed. The average of three independent experiments is shown. Error bars indicate SEM.
Schematic summary of experimental results.Figure 6A: Both myosin-mediated contraction and actin-generated protrusion result in flow of the cytoskeleton and inward directed traction force. B-D: Schematic representation of the actin (red) and myosin distribution (green), along with the resulting traction force direction (blue arrows with size indicating magnitude) for the three different modes. Shown here are the two different fan-shaped morphologies (B), and the oscillatory (C) and amoeboid cell (D) at three distinct times.
Figure 1(A) Four lung cancer data sets including TCGA_LUAD database, and GSE40791, GSE75037, GSE7670 from GEO database were intersected with human RBP database ATtRACT to identify the differentially expressed RBPs in LUAD. The Venn diagram depicts the overlap between 5 independent data sets. (B) The upper panel showing qRT-PCR analysis of four identified LUAD-associated RBPs (ADARB1, CELF2, QKI and ZFP36) mRNA levels in 61 LUAD tissues and matched paracarcinoma tissues. The lower panel showing relative mRNA expression (T/N) of the four RBPs in metastatic (n = 28) and non-metastatic (n = 33) LUAD tissues. LUAD tissues were classified into metastatic and non-metastatic tissues as described in Materials and Methods. T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression (T/N) of four RBPs. (C and D) Comparison of QKI mRNA and protein expression between normal lung tissues and LUAD tissues, which were stratified by clinical stages (Stages 1-4) in TCGA database. Data are shown as the boxplot. Central band: median; Upper and lower line of boxes: upper and lower quartile; Upper and lower line of whiskers: maximum and minimum. (E-G) Kaplan-Meier survival curves of LC, LUAD and LUSC patients (n=1540, n=865 and n=675, respectively) with high or low expression levels of QKI. The log-rank test was used to analyze the difference between two groups. (H) qRT-PCR analysis of endogenous mRNA levels of QKI-5, QKI-6, and QKI-7 in human bronchial epithelial cell line BEAS-2B, LUAD cell lines A549 and H1299. (I) Western blot analysis of QKI-5 protein levels in BEAS-2B, A549 and H1299 cells. β-actin was used as an internal control. (J and K) qRT-PCR analysis of QKI-5 mRNA in 61 LUAD tissues and matched paracarcinoma tissues (J) and T/N mRNA expression of QKI-5 in 61 paired LUAD tissues (K). T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression of QKI-5. In the violin plot, central dotted line represents median, upper and lower dotted line represents quartile (J). (L) Violin plots showing QKI-5 mRNA expression (T/N) in metastatic (n = 28) and non-metastatic (n = 33) LUAD tissues. Central dotted line of the violin plot represents median, and upper and lower dotted line represents quartile.
Destabilizing mutations in DHFR induce filamentous phenotype due to loss of DHFR activity.Figure 1Growth rates of mutant DHFR strains at 30°C, 37°C and 42°C. While most mutants grow well at 30°C, they grow very poorly at high temperatures. Error bars represent SEM of three biological replicates. Live cells DIC images and DAPI nucleoids staining of WT DHFR and I91L+W133V DHFR strains after being grown at 30°C, 37°C, or 42°C (I91L+W133V was grown at 40°C) in M9 medium supplemented with amino acids for 4 hours (see Methods). Cell lengths were measured from the obtained DIC images (see Methods) and their distribution at 37°C and 40°C/42°C is shown in (C) and (D) as box-plots (see Methods). Images of other mutant DHFR strains W133V, V75H+I155A and V75H+I91L+I155A are presented in related Figure EV1A. Distribution of cell lengths of WT and mutants W133V and V75H+I155A at 42°C (gray box) and I91L+W133V at 40°C (represented as white box) after being grown in M9 minimal medium (without amino acids) for 4 hours. Median cell length of W133V and V75H+I155A is significantly smaller than WT (Mann-Whitney test, p-value <0.001). Functional complementation of WT DHFR from an arabinose inducible pBAD plasmid rescues filamentation of mutant strains grown at 42°C (for WT, W133V and V75H+I155A strains) or at 40°C (for I91L+W133V and V75H+I91L+I155A) in M9 medium supplemented with amino acids. For the boxplots in panels , F the central band represents the median of the distribution, the box ends represent the 25th and 75th percentile, the whiskers represent the 10th and 90th percentile, while the dots represent the 5th and 95th percentile. Data was usually obtained from 2-3 biological replicates. The number of cells used to derive the boxplot distributions in the different panels range usually between 200 to 450 (please refer to Figure 1 source data for exact number of cells for each dataset). See related Figure EV1B. Functional complementation of WT DHFR from an arabinose inducible pBAD plasmid rescues growth defects of mutant strains grown at 42°C (for WT, W133V and V75H+I155A strains) or at 40°C (for I91L+W133V and V75H+I91L+I155A) in M9 medium supplemented with amino acids. Expression of mutant proteins from pBAD plasmid on the WT background does not result in filamentation For the boxplots in panels the central band represents the median of the distribution, the box ends represent the 25th and 75th percentile, the whiskers represent the 10th and 90th percentile, while the dots represent the 5th and 95th percentile. Data was usually obtained from 2-3 biological replicates. The number of cells used to derive the boxplot distributions in the different panels range usually between 200 to 450 (please refer to Figure 1 source data for exact number of cells for each dataset). See related Figure EV1B. Expression of mutant proteins from pBAD plasmid on the WT background does not result in growth defects.
Figure 7Iterative deletions using the MinGenome algorithm. Faded circles represent genome size and bright triangles the number of deleted coding and non-coding genes. Low, intermediate, and optimal growth rate constraints correspond to 60, 90, and 100% of the M. florum growth rate measured in CSY (0.44 hr-1). Venn diagram showing the protein coding genes shared between M. florum (red circle), JCVI-syn3.0 (green circle), and its parent JCVI-syn1.0 (blue circle). The different shades of grey represent the proteins targeted for deletions in the Low growth rate constraint presented in a. Voronoi diagram showing the functional distribution of 145 proteins targeted for deletions present in JCVI-syn3.0 (black), M. florum only (dark grey), and JCVI-syn1.0 but not JCVI-syn3.0 (light grey). The shapes are sized according to transcriptomic data and the KEGG categories are represented by bright colors. The single protein associated with environment information processing is represented in black on the top part of the diagram between the genetic information processing and metabolism categories. Voronoi diagram showing the functional distribution of the retained proteins in the predicted reduced genome. Shape colors are as in b, and purple represents proteins shared with JCVI-syn3A but absent in JCVI-syn3.0. Section borders are colored according to KEGG categories depicted in c.
Figure 6During anterograde IFT, kinesin-II carries IFT-A, IFT-B and inactive IFT dynein from ciliary base to tip. Inactive dynein is activated at ciliary tip and carries IFT-A and IFT-B back for retrograde transport while kinesin-II diffuses. IFT54 interacts with both kinesin-II and IFT dynein during anterograde IFT. Deletion of residues 261-275 of IFT54 disrupts its interaction with IFT dynein leading to inhibition of anterograde transport of IFT dynein and accumulation of IFT complexes near the ciliary tip with bulge formation. In contrast, deletion of residues 342-356 of IFT54 strengthens its interaction with kinesin-II, supposedly inhibiting motility of kinesin-II via unknown mechanism leading to cessation of IFT shortly after IFT trains enter cilia. IFT complexes and motors thus accumulate at proximal end of cilia with bulge formation. Please note, Δ342-356 mutant shows stronger cilia mutant type relative to Δ261-275 mutant.
Figure 1(A) UHMK1 levels in tumor tissues (T) and corresponding nontumor tissues (N) from five GC patients were analyzed using Western blotting and qRT-PCR. The relative mRNA expression of UHMK1 was presented as mean ± standard deviation from three replicates. Pairwise, two-tailed statistical significance was assessed by Student's t‐test. Data information: *P< 0.05, **P< 0.01, ***P< 0.001. (B) Immunoblotting and qRT-PCR were used to measure the levels of UHMK1 in GES-1 and GC cell lines. The relative mRNA expression of UHMK1 was presented as mean ± standard deviation from three replicates. Pairwise, two-tailed statistical significance was assessed by Student's t‐test. Data information: *P< 0.05, **P< 0.01, ***P< 0.001. (C) IHC analysis of UHMK1 expression in a GC patient tissue array (n=102). Representative images are shown. Scale bars=100μm. (D) The IHC signals were scored using the Allred score Data were shown as mean ± standard deviation from three independent experiments. Data information: *P< 0.05, **P< 0.01, ***P< 0.001. (E) UHMK1 expression in 100 GC patients was analyzed based on the following parameters: tumor stage, tumor status, and lymph node invasion status. Fisher's test was applied to calculate the exact P value. Data information: *P< 0.05, **P< 0.01, ***P< 0.001. (F) Patients with GC were stratified by the UHMK1 level (n=100); OS and DFS were assessed by Kaplan-Meier analysis. Data information: *P< 0.05, **P< 0.01, ***P< 0.001.
The LC3 protein subfamily is essential for autophagy.Figure 1(A) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of LC3 siRNAs (A, B, C), using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer. (B) Quantification of relative p62 and GFP‐GATE‐16 level (upper panel) and degradation (lower panel) was performed as described in 'Materials and methods'. Mean±s.d. of three independent experiments is presented. (C) HeLa cells stably expressing GFP‐GATE‐16 were transfected with non‐targeting siRNA or a pool of LC3 siRNAs and treated as in (A). The cells were subjected to immunostaining with anti‐p62 after fixation, and analysed by confocal microscopy. The GFP intensity of three independent experiments was measured as described in 'Materials and methods' (right panel). Scale bar: 20 μm.
Figure 1A. Schematic domain organization of Drosophila PlexA and Sema1b (SS, signal sequence; TM, transmembrane region, red symbols represent the position of N-linked glycosylation sites) B, C. Ribbon representation of the PlexA1-4-Sema1b1-2 1:1 (C) and 2:2 (D) complexes. N-glycans are shown in stick representation. D. Ribbon representation of the side-on orientation derived from the 2:2 complex. E. Microscale thermophoresis binding experiment for PlexA1-4-mVenus and Sema1becto (red) or Sema1becto-mutA (blue) or Sema1becto-mutB (green,) or Sema1becto-mutA+B (orange). The Sema1becto wild-type binding results are as reported previously (Rozbesky et al., 2019). All data were collected at the same time, error bars represent s.d. of three technical replicates.
Exome-sequencing analysis.Figure 5Number of somatic point mutations identified in primary tumors and new mutations acquired in the orthoxenograft models in PRS-WCR. Somatic mutations found in primary tumors in sporadic cases were maintained in the orthoxenograft-derived tumors, whereas few acquired mutations were observed in all NF1-derived models. PT, primary tumor; OT, orthotopic xenograft tumor; passage 1 (P1); passage 4 (P4).
The GBS pigment induces NLRP3 inflammasome-dependent cell death in human macrophages.Figure 4A, B THP-1macrophages transfected with empty vector, scrambled control, shASC, or shNLRP3 were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by Luminex assay (B). Data from three independent experiments performed in triplicate are shown.Data information: Data were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM. (A) n = 3, for WT: **P = 0.001 (vector versus shNLRP3), **P = 0.0016 (vector versus shASC); for ΔcovR: ***P = 0.0008 (vector versus shNLRP3), ***P = 0.0001 (vector versus shASC), *P = 0.047 (scrambled versus shNLRP3), **P = 0.0054 (scrambled versus shASC). Data obtained from THP-1/vector was not significantly different from THP-1/scrambled, P = 0.18. (B) n = 3, *P = 0.025. Data obtained from THP-1/vector were not significantly different from THP-1/scrambled, P = 0.89. C, D The shRNA THP-1macrophages were incubated with various concentrations of GBSpigment or ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release in supernatants was measured by Luminex assay (D). Pigment-mediated cytotoxicity is dependent on the NLRP3 inflammasome components, suggesting that pigment is inducing a programmed cell death. Data shown are the average of at least three independent experiments performed in triplicate.Data information: Data were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM. (C) n = 3, for 4 μM pigment: **P = 0.0063 (vector versus shNLRP3), **P = 0.0035 (vector versus shASC), *P = 0.01 (scrambled versus shNLRP3), **P = 0.009 (scrambled versus shASC); for 2 μM pigment: **P = 0.0088 (vector versus shNLRP3), **P = 0.0066 (vector versus shASC), *P = 0.01 (scrambled versus shNLRP3 or shASC); for 1 μM pigment: *P = 0.02 (vector versus shNLRP3), *P = 0.01 (vector versus shASC). Data obtained from THP-1/vector were not significantly different from THP-1/scrambled, P = 0.99. (D) n = 3, ****P < 0.0001, *P = 0.015.
SARS-CoV-2 N LLPS is modulated by salt and RNA.Figure 4Phase separation over time as monitored by turbidity of 50 μM MBP-N in 50 mM Tris pH 7.4 after addition of TEV protease A) with varying torula yeast RNA (at 100 mM NaCl) or B) varying NaCl concentrations (at constant RNA concentration). Error bars represent standard deviation of three replicates. DIC micrographs of 50 μM MBP-N in 50 mM Tris pH 7.4 C) with varying torula yeast RNA concentrations (at 100 mM sodium chloride) and D) varying sodium chloride concentrations (at constant RNA concentration), with or without TEV protease (to cleave MBP from N). Scale bars represent 50 μm.