EMBO/sd-character-level-ner huggingface dataset:
Figure 1A Expression of RBM47 was induced by DENV infection in 293T, HFF, HUVEC, and THP-1 cells. Cells were infected with DENV-2 at an MOI of 1 for 12 or 24 h. The expression of RBM47 mRNA (12 and 24 h post infection, hpi.) and protein (24 hpi.) were compared between virus-infected and non-infected cells (PBS). The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: Data are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). B, C Both mRNA and protein levels of RBM47 were elevated in SeV-infected (B) or IFN-α-stimulated (C) 293T, HFF, HUVEC, and THP-1 cells. Cells were infected with SeV (MOI = 1) or stimulated with IFN-α for 6 or 12 h, and the expression of RBM47 mRNA (at 6 and 12 h post treatment) and protein (at 6 and 12 h post treatment) were tested. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: Data are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). D Three putative STAT1 binding motifs in the RBM47 gene promoter region as predicted by using JASPAR (http://jaspar.genereg.net/). E The pGL3-RBM47 promoter plasmid (pGL3-47) or the empty vector was transfected into 293T cells, and RBM47 promoter activities were tested upon SeV infection or IFN-α stimulation.Data are represented as the means ± SD of n = 3 biological replicates. Data information: Data are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).
Figure 2A) mRNA fold-changes of Id1, endogenous (mouse) and transgenic (human) Zrf1, pluripotent markers Cdh1 and Oct4, the neural markers Pax6, Sox1, Olig2, HoxB1 and the epiblast/mesodermal marker T in control ESCs, Id1-Flag ESCs, or Id1-Flag ESCs transiently over-expressing Zrf1. Self-renewing conditions (d0), and day 3 and day 5 of neural differentiation were analyzed. SD is representative of three independent experiments. *P < 0.05 and ** P < 0.01 (paired t-test) represent comparison with Ctrl ESCs at the corresponding time point. B) ChIP analysis for ZRF1 in control ESCs, Id1-Flag ESCs and Id1-Flag ESCs transiently over-expressing Zrf1, grown in self-renewing conditions (d0) and during neural differentiation (day 5). SD is representative of three independent experiments. Dashed line represents IgG background. ** P < 0.01 (paired t-test). C) Immunostaining of the neural progenitor marker Nestin and of the post-mitotic neuronal marker b3 tubulin in control ESCs, Id1-Flag ESCs and Id1-Flag ESCs transiently over-expressing Zrf1 at day 5 of neural differentiation.
Traction force maps and distributions of signaling components in fan shaped cells.Figure 2A: Snapshots of LimE-GFP, GFP-myo, and corresponding kymographs for type 1 cells. Corresponding speeds are 10.8µm/min and 9.4µm/min. White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint, as extracted from the corresponding stress kymographs. C: Snapshots of LimE-GFP, GFP-myo, and corresponding kymographs for type 2 cells. Corresponding speeds are 5.6µm/min and 8.7µm/min. White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint. B: Stress maps quantifying the magnitude of the force per area using a color scale with blue/red corresponding to small/large stresses and the direction of forces using vectors. Fan-shaped cells were rotated so that the vertical (x) axis is the direction of motion (see Methods). Shown are the overall stress T, the stress in the direction of motion Tx and the stress perpendicular to motion Ty for the type 1 cell with the LimE marker, and the corresponding Tx and Ty kymographs along the cells outlines. D: Stress maps of T, Tx and Ty for the type 2 cell with the LimE marker, and corresponding Tx and Ty kymographs along the cells outlines. E: Left three panels: Major and minor axis of the cells, Lmaj and Lmin, and total force F as a function of the cell area A. Right panel: Force perpendicular to the motion, Fy, as a function of the force parallel to the motion, Fx. The dashed line represents a linear fit with a slope of 1.87 (r2=0.88). The plots present averaged values for each cell based on the duration of each recording (cell type 1: blue markers, cell type 2: orange markers, less stable cells: gray markers (see Methods)). The basal area and speed of type 2 cells was larger than for type 1 cells: 628 (502/692) μm2 and 6.0 (5.4/8.2) μm/min (N=12 biological replicates) vs. 326 (258/461) μm2 and 10.8 (9.4/12.3) μm/min (N=161 biological replicates) (p=1.9x10-6 and 2.6x10-7) while the median ratio between the pole-pole distance and the cell's length was 0.75 (0.70/0.79, N=161 biological replicates) for type 1 cells and 0.84 (0.77/0.90, N=12 biological replicates) for type 2 cells (p=2.2 10-3).
Electron microscopy of the ASPC-1 and MiaPaCa-2 cell lines treated or untreated with OMP.Figure 4(A) ASPC-1 cell without treatment (800 fold). (B) ASPC-1 cell undergoing apoptosis upon 160 µg/ml OMP after 24 hours (800 fold). Vacuolisation of the cytoplasma and condensation of the nucleus are visible. (C) Phagophores and autophagosomes in a segment of an ASPC-1 cell treated with omeprazole 80 µg/ml for 24 hours (2800fold enlargement). The phagophores are characterised by a cup-like shape (white arrows). Autophagosomes are closed particles, the number of which is increased in treated cells (black arrows). (D) Early phagophores and autophagosomes are also found in MiaPaCa-2 cells treated with OMP 80 µg/ml after 24 hours in a perinuclear region containing lysosomes and the Golgi complex. In contrast to ASPC-1 cells, early signs of apoptosis such as vacuolization, are also present. (E) Barchart of the numbers of autophagosomes and lysosomes per cell in MiaPaCa-2 and ASPC-1 cells untreated or treated with 5-FU, OMP or the combination of both for 24 hours with standard errors. Significant differences compared to control are marked by *. In ASPC-1 cells there were significant differences compared to the control in the OMP group (p: 0.03) and the 5-FU+OMP group (p: 0.03). In MiaPaCa-2 cells the 5-FU+OMP group differed significantly from control (p<0.001).
Effect of inhibitors on transport of peroxisomal GFP-SKL to, and degradation in, vacuoles during (a) glucose adaptation and (b) ethanol adaptation.Figure 6Effect of inhibitors on transport of peroxisomal GFP-SKL to, and degradation in, vacuoles during (a) glucose adaptation and (b) ethanol adaptation. Wortmannin and PMSF were dissolved in DMSO, and added to the glucose medium. (Filled circle) Control (2% DMSO); (open circle) 1 mM wortmannin (with 2% DMSO); (filled square) 3 mM PMSF (with 2% DMSO); (filled triangle) 10 mg/ml cycloheximide.
Atg1–spaghetti‐squash activator (Sqa)‐mediated myosin II activation is required for starvation‐induced autophagy.Figure 4(A, B) Activation of myosin II on nutrient deprivation. The larval fat body of denoted genotypes under fed or starved conditions were dissected, lysed, and subjected to western blot analysis using antibodies specific for phospho‐myosin regulatory light chain (MRLC) and total MRLC. The Rheb, SqaRNAi, Sqa‐T279A, spaghetti‐squash (SqhA20A21), and Atg7RNAi transgenes were expressed under the control of hs-GAL4 driver (B). For quantification, the relative phosphorylation levels of MRLC were quantified as in Figure 3D. Data are represented as mean±s.e. of triplicates. (C-J) Starvation‐induced autophagosome formation was compromised by inhibition of myosin II activation. Compared with the fed condition (C), starvation induced a robust formation of GFP-Atg8a puncta in larval fat‐body cells (D). Clonal expression of Atg12RNAi (E), Sqa‐T279A (F), SqaRNAi (G), or SqhA20A21 (H) markedly suppressed GFP-Atg8a puncta formation during starvation. The autophagic defects caused by Sqa‐T279A and SqaRNAi were rescued by co‐expression of the constitutively active SqhE20E21 (I) and SqhD20D21 (J), respectively. Bar, 20 μm. (K) The average number of GFP-Atg8a marked autophagosomes per cell is shown (data are represented as mean±s.e. of 20 fat‐body samples imaged per genotype; ***P0.001). (L) Inhibition of myosin II activity increased sensitivity to starvation. Flies carrying transgenes under the control of fat body‐specific Cg-GAL4 driver were used for further analysis. Histogram illustrating the survival curve of adult female flies of denoted genotypes when placed under starved conditions. Data are mean±s.e. from triplicate experiments (n=100 flies/genotype/treatment). *P0.05, **P0.01, ***P0.001. See Supplementary data for genotypes.
Schematic representation of miR-27a/24 in silencing ESC self-renewal and promoting differentiation.Figure 8Figure 8 Schematic representation of miR-27a/24 in silencing ESC self-renewal and promoting differentiation.
Figure 4(A) Close-up of the TRIM25 RING dimer interface highlighting the hydrophobic interactions made between the four helices (left). Close-up of the interface between each RING monomer and the proximal ubiquitin (right). (B) UBE2D1~UbAtto discharge assays with TRIM25 wild-type RING, the fused RING constructs and different mutants important for dimerization or the interaction with ubiquitin. Time point zero for the T25-R Linker and T25-R Linker V72R samples was taken before the addition of E3 as discharge is very fast. The loss of UBE2D1~UbAtto is plotted as the average of experimental duplicates (± s.d.). (C) UBE2D3~Ub discharge assays with the same mutants as in (B), stained with InstantBlue. (D) K63 poly-ubiquitination assays using UBE2N/UBE2V1. The asterisk indicates the band for the TRIM construct.
tumours (A) Significantly deregulated canonical pathways in Cx3cr1-Rheb1Δ/Δ versus Rheb1fl/fl GL261 tumours, as identified by the IPA software from differentially regulated genes and divided in three categories: Adaptive immunity, Cytokine signalling, and Innate immunity.Figure 4(A) Significantly deregulated canonical pathways in Cx3cr1-Rheb1Δ/Δ versus Rheb1fl/fl GL261 tumours, as identified by the IPA software from differentially regulated genes and divided in three categories: Adaptive immunity, Cytokine signalling, and Innate immunity. Size of bubbles is indicative of ratio of differentially regulated genes. The x-axis represents -log10(p-value) with a threshold of p≤0.05 applied. (B) Levels of TAM-MG (P2RY12+ CD49d-) and TAM-BMDM (P2RY12- CD49d+) gated from CD45+ population in GL261 tumours from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=5) mice by flow cytometry (representative flow cytometry plots on top and quantification at the bottom; mean ±SEM; Two-Way ANOVA Tukey test). (C) Percentage of CD8 CTL (top) and CD4 Th (CD4) cells in GL261 tumours from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=5) mice assessed by flow cytometry (representative flow cytometry plots, left and quantification, right; mean ±SEM; Unpaired t test). (D) Tumour tissues from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=4) were stained for CD8, CD4 and FoxP3. DAB staining was quantified as % of positive cells using Definiens software. Representative images of the stained tissues are shown. (mean ±SEM; unpaired parametric t test). Scale bar is 100µm.
Figure 2(A) Heat map visualization of top 30 differentially regulated mRNA transcripts. Color represents significant down- (blue) or up-regulated (red) mRNA expressed as a log2 fold change relative to vehicle corrected for the false discovery rate. (B) Schematic illustration of differentially regulated mRNA transcripts involved in the AMPK signaling pathway. Structures highlighted in shades of red were differentially regulated by BAM15. Darker shades of red correlate to increased probability of activation. Structures highlighted in grey or with black borders were unaltered by BAM15. (C) Pathway enrichment analysis of differentially regulated mRNA transcripts.
SNX18 binding to PI(4,5)P2 membranes is required for its function in autophagy.Figure 4.(A) HeLa GFP-LC3 cells were transfected to express myc-SNX18, immunostained against myc, and analyzed by confocal imaging. Asterisks indicate a nontransfected cell. Bars: (main panels) 20 µm; (inset) 10 µm. (B) HeLa cells transfected with flag-Atg16L1 and myc-SNX18 were starved or not starved for 2 h before immunostaining with anti-myc and anti-flag antibodies and confocal microscopy. Bars, 5 µm. (C) SNX18 domain structure. An N-terminal SH3 domain is followed by a low-complexity region (LC). The membrane-remodeling part consists of an amphipathic helix (helix 0), a PX domain that is flanked by sequences forming a Yoke domain (Y), and a BAR half-domain. The functional BAR domain is formed by dimerization. Truncated constructs and point mutations used are indicated. (D) HeLa cells were transfected with the indicated myc-SNX18 plasmids for 6 h and starved for an additional 2 h before harvest. Equal parts of cytosol (C) and membrane (M) fractions were analyzed by Western blotting. The graph shows the average membrane-bound myc-SNX18 ± SEM (error bars), n = 3. (E and F) HEK GFP-LC3 cells were transfected with the indicated mCherry-SNX18 (E) or mycSNX18 (F) constructs for 16 h. The number of GFP-LC3 spots per transfected cell (E; graph shows mean ± SEM [error bars], n = 5) or ratio of LC3-II to actin determined by Western blotting (F) was quantified. (E and F) HEK GFP-LC3 cells were transfected with the indicated mCherry-SNX18 (E) or mycSNX18 (F) constructs for 16 h. The number of GFP-LC3 spots per transfected cell (E; graph shows mean ± SEM [error bars], n = 5) or ratio of LC3-II to actin determined by Western blotting (F) was quantified. (G and H) HEK GFP-LC3 cells transfected with myc-SNX18 or a myc control plasmid alone (G) or together with CFP-FRB and mRFP-FKBP or mRFP-FKBP 5pase (H) for 6 h were treated or not treated with 5 µM ionomycin (G) or 2.5 µM A/C heterodimerizer (rapalog; H) for the last 3 h before analysis of the total intensity of GFP-LC3 spots per transfected cell (graph shows mean ± SEM [error bars], n = 3). (I) MEFs were transfected with myc, myc-SNX18 WT, or KR for 16 h, starved or not starved for 2 h, and immunostained for endogenous Atg16L1. Insets show enlarged views of the boxed regions. Bars, 5 µm. (J) HeLa cells were transfected for 12 h with the indicated constructs and starved for 90 min. Cells were solubilized and immunoprecipitated with anti-flag or an unrelated antibody of the same isotype (mock ab). Immunoprecipitates and 2% of the lysates were analyzed by immunoblotting against myc and flag. *, P < 0.05; **, P < 0.01. See also Fig. S3.
Figure 10(A, B) The nucleolin GAR domain binds a kinesin light chain, directly linking nucleolin-mRNA complexes to kinesin motors for axonal transport (A). The GAR domain further mediates membrane association of nucleolin. GAR-mediated subcellular targeting of nucleolin complexes enables export of key mRNAs to the axon, and the local translation of their encoded proteins for local functions in the axon, or for retrograde transport to the cell body. This reciprocal transport mechanism provides intrinsic regulation of axon length and growth, and indeed deletion or mutation of the GAR domain (B) perturbs mRNA localization to axons and increases axonal elongation.
Figure 6(a, b). Drp1 rescues PINK1KO induced collapsed thorax phenotype in dATG7-deficient Drosophila. Representative SEM images of thorax from PINK1WT, (upper panels) and PINK1KO (lower panels) flies combined with dATP7 deficiency (dATG7KO) or their controls (dATG7WT), followed by expressing either mhc-gal4 (Mhc/+) alone, or Mhc-gal4 driven expression of human Drp1 (Mhc>hDrp1WT), mhc-gal4 driven expression of human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven expression of human Drp1S616D (Mhc>hDrp1S616D) are shown (a). Quantitation of crushed thorax in flies with indicated genotypes is presented (b). > 100 flies for each experiment are analyzed. Scale bar=200µm. One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments. (c, d) Drp1 rescues PINK1KO induced mitochondrial defects in dATG7KO Drosophila. Representative TEM images of indirect flight muscle from PINK1WT (upper panels) and PINK1KO (lower panels) flies combined with dATG7KO or their controls (dATG7WT), followed by expressing either mhc-gal4 (Mhc/+) alone, or Mhc-gal4 driven expression of human Drp1(Mhc>hDrp1WT), mhc-gal4 driven expression of human Drp1S616A(Mhc>hDrp1S616A) or mhc-gal4 driven expression of human Drp1S616D(Mhc>hDrp1S616D) are shown (c). Scale bar=1 μm. Normal and abnormal mitochondrial number per mm2 from each experimental group were and quantified. >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. Bars represent the mean ± SEM of three independent experiments (d). (e). Drp1 rescues PINK1KO induced ATP reduction in Drosophila. ATP contents of thorax muscle tissues from the indicated genotypes were measured and normalized against the protein levels One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.
FAM83D recruits CK1α to the spindle: A: STLC-synchronised mitotic (M) wild-type (WT), FAM83D-/- knockout (KO) and FAM83DGFP/GFP knockin (KI) U2OS cells were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy.Figure 2A: STLC-synchronised mitotic (M) wild-type (WT), FAM83D-/- knockout (KO) and FAM83DGFP/GFP knockin (KI) U2OS cells were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µm. B: STLC-synchronised mitotic (M) FAM83D-/- knockout (KO), FAM83DGFP/GFP knockin (KI) and FAM83DGFP/GFP(F283A) knockin (FA) U2OS cells were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µ C: Quantification of CK1α spindle localisation for the cells Cell images denote the measured regions used to calculate the ratios on the box plot. Box plot whiskers denote the minimum and maximum measured values. The middle line represents the median, and the box ranges depict the 25th/75th percentiles. ***, P<0.0001; ANOVA. Analysis was performed on the indicated number of cells, n=2. D: The cell lines were STLC-synchronised and mitotic cells (M) isolated by shake off. Asynchronous (AS) cells were included as a control. Cells were lysed and subjected to GFP TRAP immunoprecipitation (IP) and subsequent immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments. E: Schematic illustration of the AdPROM-mediated degradation of FAM83D. VHL; Von Hippel Lindau protein, CUL2; cullin 2, RBX1; RING-box protein 1, E2; E2 ubiquitin-conjugating enzyme, aGFP.16; anti-GFP.16 nanobody. F: KI cells were infected with retroviruses encoding either VHL, aGFP.16, or VHL-aGFP.16. Uninfected cells were used as a control. Cells were lysed and subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments. G: The cell lines were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µm.
Figure 5Expression levels of ISGs (A) upon HNRNPC silencing in the cells with lentiviral shRNA-mediated stable knock-down of DDX58 or IFIH1. Each sample has 3 replicates. Error bars represent mean ± SD. Expression levels of IFNβ concentration in the media (B) upon HNRNPC silencing in the cells with lentiviral shRNA-mediated stable knock-down of DDX58 or IFIH1. Each sample has 3 replicates. Error bars represent mean ± SD. Growth curves of the DDX58- and IFIH1-deficient MCF7 (left) and T47D (right) cells upon HNRNPC knock-down. Each sample has 3 replicates. Error bars represent mean ± SD. Xenograft tumor models with DDX58-deficient MCF7 cells. Images, weights, and growth records of the xenograft tumor models in female NSG mice established from the DDX58-deficient MCF7 cells upon lentivirus-mediated stable knock-down of LMNA or HNRNPC. Each group has 6 mice. The error bars represent the ± SEM. Xenograft tumor models with DDX58-deficient MCF7 cells. Images, weights, and growth records of the xenograft tumors derived from DDX58-deficient MCF7 cells, which were subjected to periodic siRNA injection, starting from 2 weeks after transplantation of the cells. Each group has 4 mice. The error bars represent the ± SEM.
Spatial gene expression mapping of diencephalic astrocyte clustersA-Q Mapping of the single cell data shows different distribution of the respective clusters.Figure 2Mapping of the single cell data shows different distribution of the respective clusters. Astrocyte clusters 0 - 4, 9 and 10 are shown in panels A - E and J, K, respectively. Data information: Color-coding bars represent degree of gene expression similarity between all cells from given cluster and mixture of all cells from given single pixel of Slide-seq data. Scale bars: 300 µm Mapping of the single cell data shows different distribution of the respective clusters. Data information: Color-coding bars represent degree of gene expression similarity between all cells from given cluster and mixture of all cells from given single pixel of Slide-seq data. Scale bars: 300 µm R Schematic of mouse coronal brain section. CP: caudatoputamen; CTX: cerebral cortex; HC: hippocamus; HY: hypothalamus; int: internal capsule; LV: lateral ventricle, TH: thalamus. Data information: Color-coding bars represent degree of gene expression similarity between all cells from given cluster and mixture of all cells from given single pixel of Slide-seq data. Scale bars: 300 µm
Figure 6iPS cells in G2 express high levels of Cyclin A and do not incorporate EdU (marked in red within the NICD panel). EdU incorporation occurs during S phase (marked by yellow in the NICD panel). Cells with low Cyclin A intensity and no EdU incorporation are either in G1 or M phase. M phase cells are distinguishable by their chromatin condensation, observable in the DAPI panel (dividing cells are indicated with white in the NICD panel). G1 cells do not have any of the features described above (blue in NICD panel). Scale bars are 20 µm. G1 and G2 phase cells have significantly higher NICD protein levels than S and M stage cells (p≤0.001). All groups were compared against each other with a one-way ANOVA analysis, followed by Turkey test. M phase cells have the lowest NICD. Each point in the box-plot represents the normalized intensity of a single cell. Data from three different experiments (n=3) was randomised collated and plotted.
Figure 3(A) Western blot analysis of individual OXPHOS subunits in heart extracts. HSC70 and Calnexin are used as loading controls, (n = 3). Antibodies against individual OXPHOS subunits (on the right) were used to detect OXPHOS complexes (on the left). (B) BN-PAGE and subsequent Western blot analysis for OXPHOS complexes. Antibodies against individual OXPHOS subunits (on the right) were used to detect OXPHOS complexes (on the left). (C, D) In gel activity of complexes I (C) and IV (D) performed after BN-PAGE. (E) Oxygen consumption rates in intact heart mitochondria in the presence of pyruvate-glutamate-malate (C I) and pyruvate-glutamate-malate + succinate (C I + C II) as substrates. State 3 (substrates+ADP); State 4 (+oligomycin); Uncoupled (+FCCP). (n = 3). Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001).