EMBO/sd-character-level-ner huggingface dataset:
miR-29a expression is not experience-dependent.Figure 2Mature miR-29a expression (miR29) in visually deprived mice: a) normal rearing (NR, N=5) vs dark rearing (DRb, N=5); t-test, p=0.19, b) NR (N=4) vs dark rearing from P10 (DRp10, N=5; t-test, p=0.70. In both experiments, analysis was performed at P25 Mature miR-29a expression (miR29) in visually deprived mice: c) one week period of dark rearing beginning at P30 (DRw) followed by a 4 hour exposure to light (DRw+4h light) vs control animals (ctr, N=4 for each group; one-way ANOVA, p=0.68) Mature miR-29a expression (miR29) in visually deprived mice: d) days of MD beginning at P25, contralateral cortex vs ipsilateral cortex to deprived eye, N=5; paired t test, p=0.52). Error bars represent ±SEM, symbols represent individual cases.
Figure 6A Luciferase activity in 293T cells transfected with an ISRE (left) or IFN-β (right) promoter-driven luciferase reporter and together with plasmid encoding RIG-I (N) or cGAS and STING. B Protein lysates of WT and USP19 KO A549 cells infected with SeV at indicated time points were immunoblotted with indicated antibodies. C RT-PCR analysis of gene transcription in WT and USP19 KO A549 cells after infection with SeV at indicated time points. D Phase-contrast microscopy (PH) and fluorescence microscopy images of WT and USP19 KO A549 cells infected with VSV-eGFP at an MOI of 0.01 for the indicated time length. Scale bar, 50 μm. E Human PBMCs were transfected with control or USP19-specific siRNA, followed by treatment with influenza A/Puerto Rico/8/34 (H1N1) (PR8) (MOI = 5) at different time points, the lysates were analyzed with each antibody. F Human PBMCs were transfected with control or USP19-specific siRNA were infected with H1N1 virus (MOI = 5) at different time points. Relative expression levels of IFNB, ISG15, ISG54, ISG56 mRNA and H1N1 nucleoprotein (NP) RNA were measured by real-time PCR. G Relative expression levels of IFNB mRNA and interferon-stimulated genes, including ISG54 and ISG56 in WT, BECN1, ATG5 and SQSTM1 KO cells with or without USP19. H, I Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE (H) or IFN-β (I) promoter-driven luciferase reporter after infection with SeV for indicated time points.
Figure 6Transcriptome of female gametocyte by RNA-seq between DFsc7 (WT) and DFsc7;Δap2-o3 (KO) strains. The genes were ranked by transcripts level (FPKM average of three sample replicates) in the heatmap. In spite of global upregulation of vast genes with low or medium transcript levels (left panel), the highly expressed genes (FPKM>1500, right panel) are downregulated in KO over WT. The FPKM values in each row (each gene) were normalized by Z-score normalization. Gene percentage of downregulation and upregulation in populations of the highly expressed genes (n=111, FPKM>1500) and the others (n=4589, FPKM<1500) between KO and WT. Line chart of 50 representative highly expressed female genes The dot is the mean value of FPKM in three repeats. qRT-PCR detecting mRNA transcript level of ten selected highly expressed genes The numbers indicate the gene ID. Western blot of GAP45 protein expression in non-activated (NAG) and activated gametocyte (AG) of 17XNL and Δap2-o3 strains. BiP as loading control.
Figure 1A Northern blots comparing GlmY and GlmZ levels in wild type strain Z8 and the ∆rapZ mutant Z28 under normal growth and GlcN6P starvation conditions. Both strains, which also carried a chromosomal glmS'-lacZ fusion, were treated with 60 μg/ml Nva-FMDP or H2O ("mock"). Samples were harvested hourly for Northern analysis and determination of β-galactosidase activity. Growth curves are shown in Appendix Fig S1. Blots were re-probed using a 5S rRNA specific probe to provide loading controls. B The purification profile of Strep-RapZ from the ∆glmS strain Z904 under GlcN6P replete and depletion conditions is shown (top). The cleared lysate (CL), flow through (FT), washing steps (W) and the elution fractions (E1-3) from StrepTactin affinity chromatography were separated on 12.5 % SDS-PAA gels and stained with Coomassie blue. Metabolites were extracted from E2 and analyzed by HILIC-MS/MS. The extracted ion chromatograms of the LC-MS analysis targeting GlcN6P (retention time 16.6 minutes) are shown below. The samples derived from purification of Strep-RapZ (panels i and ii) or Strep-KdpE (panels iii and iv) were analyzed with the SRM transition m/z 258.1 to m/z 97 in the negative ion mode. The identity of the metabolite detected in panel i was confirmed by adding chemically pure GlcN6P to a final concentration of 100 pg/μl (panel v). C SPR analysis addressing interaction of RapZ variants with GlcN6P and similar metabolites. The Strep-tagged proteins were captured onto a sensor chip and various concentrations of the respective metabolite (i.e. 100, 500, 1000, 2500 and 5000 nM) were injected using a single cycle kinetics approach.
Figure 2A. HUWE1 levels are efficiently downregulated by two different HUWE1 siRNA oligonucleotides in 8988T cells. B. Quantification of HUWE1 expression following siRNA treatment. Band intensity was quantified using ImageJ software and normalized to Tubulin loading control. The average of three experiments is shown. Error bars indicate SD. C-E. Cell cycle analyses by flow cytometry using BrdU/PI double staining show increased replication arrest in HUWE1-depleted 8988T cells in the absence of exogenous DNA damage treatment. C. Representative flow cytometry profiles of control and HUWE1-knockdown cells. R1-labeled region indicates mid and late S-phase cells (BrdU-positive, >2N DNA content), while R2-labeled region indicates S-phase arrested cells (BrdU-negative, DNA content between 2N and 4N). C-E. Cell cycle analyses by flow cytometry using BrdU/PI double staining show increased replication arrest in HUWE1-depleted 8988T cells in the absence of exogenous DNA damage treatment. C. Representative flow cytometry profiles of control and HUWE1-knockdown cells. R1-labeled region indicates mid and late S-phase cells (BrdU-positive, >2N DNA content), while R2-labeled region indicates S-phase arrested cells (BrdU-negative, DNA content between 2N and 4N). D. Quantification of S-phase cells. Percentage of cells in R1 region is shown. Bars represent the average of three independent experiments. Error bars indicate SD. P-value is 0.0014. E. Quantification of S-phase arrested cells. Bars represent the fold increase in the percentage of cells in R2 region, normalized to siControl-treated cells. The average of three independent experiments is shown. Error bars indicate SD. P-value is 0.0135. F. DNA fiber experiment showing reduced replication tract length in HUWE1-depleted HeLa cells in the absence of exogenous DNA damage treatment. The "n" numbers of fibers analyzed (pooled from two independent experiments), as well as the mean, are indicated on the graphs. G, H. Clonogenic assay showing that HUWE1 knockdown in 8988T cells are sensitive to UV (G), and HU (H). As controls, both cells transfected with non-targeting siRNA (siControl), and non- transfected cells, were used. Note that the siHUWE1#2 oligonucleotide shows a stronger downregulation of HUWE1, and confers increased sensitivity compared to siHUWE1#1. The average of nine experiments is shown. Error bars represent SEM. P-values (representing the statistical difference between the samples at the highest dose treatment) are: 4.55x10-9 (G) and 1.8x10-8 (H). I. DNA fiber experiments showing decreased replication tract length following UV-B treatment (30J/m2) in HUWE1-depleted HeLa cells. The "n" numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. P-value is 2.11x10-20.
The effect of folate deficiency on DNA damage and signal transduction pathways.Figure 4Cyclin E-expressing BJ cells grown for 7 days with and without folate.Immunoblotting with anti-phosphorylated ATM and anti-phosphorylated CHK1 antibodies. Anti-β-catenin and anti-actin antibodies were used as loading controls.Protein level quantification. Cyclin E-expressing BJ cells grown for 7 days with and without folate.Examples of nuclei with RAD51 foci following cyclin E expression (CycE) (n = 67), empty vector (n = 65), folate-free medium for 7 days (−folate) (n = 71) or oncogene expression under folate-free conditions (CycE −folate) (n = 75). Green: RAD51, blue: DAPI staining.Percent of nuclei with the indicated number of RAD51 foci (n = 66). **P < 0.01.
Figure 6A Outline of the experimental set up and treatment regimens. B 1205Lu cells were s.c. inoculated into nude mice and when tumors reached 75 mm3, mice were treated with the indicated mono- or combo-therapy for 30 days. Graphs show tumor growth following treatment by indicated drugs. Data shown are mean ± SEM of tumor volume. Vehicle and Imatinib groups, n = 10 tumors from 5 mice; Vemu and Vemu/Imatinib groups, n = 20 tumors from 10 mice). ****P<0.0001, two-way ANOVA followed by Tukey's multiple comparison. C Scatter plot graphs showing the tumor weight upon treatment by the indicated mono- or combo-therapy. Error bars show mean ± SEM of tumor weight (n = 10 to 11 tumors from 6 mice per condition). ***P=0.0002, Kruskall-Wallis test followed by Dunn's multiple comparison test; ns, non significant. D Immunofluorescence staining using anti-cleaved Caspase 3 (red), anti-Ki67 (green) and DAPI in tumor sections of 1205Lu-derived xenografts from (B). Scale bar, 100 µm. A microphotograph of the tumor size in each treated group is shown (representative of n = 5 to 10 mice). E Kaplan-Meier survival curves of mice treated with vehicle, Imatinib, BRAFi or BRAFi plus Imatinib. Median time to progression was 18, 20, 36 and 48 days, respectively. Log rank (Mantel-Cox) for BRAFi vs BRAFi/Imatinib mesylate. ****P<0.0001 and hazard ratio (log rank): 0.2403 (95% CI of ratio, 0.08123 to 0.7106). F Mouse body weight was measured at the indicated day. Data shown are mean ± SEM (n = 5 to 10 mice).
ΔPrPs spontaneously form aggregates that are detergent-insoluble and relatively chymotrypsin-resistant.Figure 3A. ΔPrPs are insoluble in detergent solution. Immunoblots showing partition of Δ159 and Δ159-175 in supernatant (Sup) and pellet fraction after ultracentrifugation of lysates supplemented with 4% sarcosyl. Upper panel is mAb 3F4, lower panel is membrane re-probed with mAb 4H11 detecting also endogenous wild-type PrP besides ΔPrPs. The majority of ΔPrPs is found in the insoluble fraction in both infected and non-infected cells (upper panel, lanes 3, 4, 7 and 8). Wild-type PrP almost completely partitions into the soluble fraction (Sup) in non-infected N2a cells with present conditions. PrP in pellet fractions of non-infected N2a cells is from ΔPrPs as can be seen from the banding pattern (Lane 7 and 8, square bracket). PrP in lanes 3 and 4 (lower panel) is PrP27-30. B. ΔPrPs are moderately resistant to chymotrypsin digestion. Immunoblots showing chymotrypsin-resistant fragments of Δ159 and Δ159-175 from transiently transfected N2a cell lysates digested with indicated concentrations of chymotrypsin (Chy). Note that full-length diglycoform of ΔPrP still remains at 8 µg/ml Chy (lanes 5 and 10, upper panel, closed arrowhead), whereas that of endogenous wild-type PrP is completely digested (lanes 5 and 10, lower panel, open arrowhead) and only small amounts of truncated fragments remained (curly bracket). Other smaller fragments in lane 5 and 10 (square-brackets) represent ΔPrPs.
Visualization of UIS4‐positive tubules in infected hepatoma cells by correlative light and electron microscopy (CLEM).Figure 7A and B) A phase‐contrast image containing the infected cell of interest (A) was used for comparison to the EM grid after fixation and preparation of the cells for TEM (B). Green outlining of the cells in the phase‐contrast image was used as a tool for comparison and alignment. The insets defined in white are shown in the right panels merged with the fluorescent image of the UIS4‐mCherry signal acquired immediately prior to fixation. Scale bars, 10 µm. C) Detection of the UIS4‐mCherry signal by widefield fluorescence microscopy in the cell of interest immediately prior to fixation. The location of the parasite is marked with an asterisk, and UIS4‐positive features are highlighted with arrowheads. D) Thirty‐six TEM sections of this sample were prepared and imaged (Movies S4 and S5). Features corresponding to the UIS4‐positive tubule (white arrowhead) were found in sections 10-16 and to the UIS4‐positive signal adjacent to the PVM (yellow arrowhead) were found in Sections 16-29. Sections 11 (top) and 20 (bottom) are displayed to highlight these two features. Scale bars, 1 µm.
Figure 1(A) Amylose pulldown from benzonase-treated HEK293T cell lysates expressing 2xMBP-BRCA2NT in untreated or irradiated cells (6Gy; +IR). DDX5 and BRCA2NT (MBP) detected by immunoblot. StainFree images of the gels before transfer were used as loading control (B) Immunoprecipitation (IP) of endogenous BRCA2 from benzonase-treated HEK293T cell lysates left untreated or treated with IR (6Gy) and harvested 4h post-IR, as indicated. Mouse IgG was used as negative control. Immunoblot of DDX5 and BRCA2. StainFree image of the gels before transfer were used as loading control (cropped image is shown). Asterisk (*) indicate a non-specific band detected by anti-DDX5 antibody. (C) Left: Representative images of in situ proximity ligation assay (PLA) between BRCA2 and DDX5 antibodies in U2OS cells either left untreated (-) or irradiated (4h post-IR; 6 Gy). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls from untreated siC cells are shown. Scale bar indicates 10 µm. Right: Quantification of the number of PLA spots per nucleus. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot. (D) Diagram showing the BRCA2 N-terminal truncations used in this study and amylose pulldown from HEK293T whole cells extracts overexpressing the indicated BRCA2 N-terminal truncations (BRCA2T1, BRCA2LT2, BRCA2LT3) or the 2xMBP tag. DDX5 and BRCA2 truncations were detected using specific antibodies against DDX5 and MBP, respectively. StainFree images of the gels before transfer were used as loading control (E) Left: GST pulldown assay using purified BRCA2T1 and DDX5; MBP antibody was used for the detection of both proteins. UB: unbound; E: eluate. Right: SDS-PAGE showing 300 ng of purified MBP-DDX5-GST and of 2xMBP-tagged BRCA2T1 used in the pulldown assay.
Figure 5A, D Stably transfected MDA-MB-231 cells bearing a control (A), DAPK1 (B), UNC5B (C), or NTN1 shRNA (D) were injected into the mammary fat pad of immuno-compromised mice. When tumors reached 100-120 mm3, mice were injected subcutaneously with DAC (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg). Tumor volumes were measured twice a week, n = 8 mice per group. The statistical significance of the differences obtained between DAC-group and DAC + net1-mAb-group for shControl, DAC-group and DAC + net1-mAb-group for shDAPK1 and shUNC5B, and PBS-group and DAC-group for shNTN1, respectively, were determined by two-way ANOVA and a post-hoc Tukey-test. **** P<0.0001, , ns = not significant. Error bars = s.e.m.
Figure 3A. Schematic of competitive BM transplantation assay. B. Percent of total donor-derived, hematopoietic cells (CD45.2+), B-cells (B220+), myeloid cells (CD11b+Gr1+), and T cells (CD3+) in the peripheral blood (PB) of recipient mice, as determined by FACS at the indicated time points. The statistical significance of differences was determined using two-ANOVAs with Holm-Sidak's multiple comparisons tests (n=8 recipients per genotype in each experiment). Data information: All data are presented as mean±SD. *p<0.05, **p<0.001, with significance determined by repeated measures of two-way ANOVA analysis with Sidak's multiple comparison tests for comparison of recipients of WT and PKCδ KO marrow at each time point and for each cell population. C. Percent donor-derived HSPCs in the BM of secondary (left) and tertiary (right) recipients of WT or PKCδ−/− marrow. Data are compiled from two independent experiments (n=8 recipients per genotype). Data information: All data are presented as mean±SD. *p<0.05, **p<0.001, with significance determined by repeated measures of two-way ANOVA analysis with Sidak's multiple comparison tests for comparison of recipients of WT and PKCδ KO marrow at each time point and for each cell population.
A GA-rich motif in the mouse Rab13 3'UTR is necessary for localization at protrusions.Figure 2A. Schematic showing the %GA content along the mouse Rab13 3'UTR using a 30nt window size. Occurrences of the consensus GA-rich motif are indicated by a red rectangle. The exact sequence between nucleotides 153-216 is shown with GA motifs in red and deleted regions indicated by black bars. P-value by Fisher's exact test with Bonferroni correction. B. FISH images of mouse fibroblasts expressing the β-globin coding sequence followed by the indicated UTRs. β-globin RNA is shown in yellow. Nuclei and cell outlines are shown in blue. Arrows points to β-globin RNA concentrated at protrusive regions. ∆1 and ∆1+2 indicate deletions of the regions shown in (A). Scale bars: 10 μm. C. Distribution of β-globin RNA, or of Ddr2 RNA detected in the same cells, quantified by measuring a Peripheral Distribution Index (PDI). N=35-55 cells observed in 3 independent experiments. Bars: mean ± 95% CI. ****:p<0.0001 by analysis of variance with Dunnett's multiple comparisons test.
Figure 4A CANBD- and CADMAC-incorporated lignin polymerization. CANBD (green) and CADMAC (blue) fluorescence was observed in the lignified infection site at 2 dpi, as revealed by phloroglucinol staining. Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site White dash lines indicate the boundary between IS and UIS. Scale bars, 500 μm B Cross-sections of CANBD- and Pst DC3000 (AvrRpm1)-treated leaves. Data information: Bacterial inoculum was at 108 cfu/ml. hpi, hours post-inoculation Scale bars, 40 μm C Cross-section of the entire leaf with one half leaf infected. Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site; hpi, hours post-inoculation Scale bars, 120 μm D CADMAC-incorporated lignin deposition in the GFP-Pst DC3000 (AvrRpt2)-infected site at 2 dpi. Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site DC3000, Pst DC3000; AvrRpt2, Pst DC3000 (AvrRpt2); Chl, chlorophyll. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μm E Cellular observation of CADMAC-incorporated lignin formation against infecting GFP-Pst DC3000 (AvrRpt2). Data information: Bacterial inoculum was at 108 cfu/ml. hpi, hours post-inoculation Scale bars, 20 μm F 3D image of CANBD-polymerized structure. Images were taken at 24 hpi Data information: Bacterial inoculum was at 108 cfu/ml. Scale bars, 20 μm
Figure 6(A) Growth curves for HCT116 recipient cells in reduced (1%) serum conditions following 30 min pre-incubation with EVs isolated by UC (104 per cell; top) or by SEC (4 x 103 per cell; bottom) from glutamine-replete and -depleted HCT116 cells or with vehicle (PBS). Fold change in confluency is a measure of cell area occupying well relative to zero time, as measured by IncuCyte software. (B) Cumulative tube length for HUVEC recipient cells following treatment with 104 EVs isolated by UC from glutamine-replete and -depleted HCT116 cells or with vehicle (PBS). Both EV preparations promote tubulation, but the network is more stable with EVs from glutamine-depleted HCT116 cells. (C) Western blot analysis of EVs isolated by UC from HCT116 cells cultured in glutamine-replete and -depleted medium for 24 h, following transduction with a Rab11a or control non-targeting (NT) shRNA knockdown construct over previous two days. Bar chart shows change in putative exosome proteins in EVs secreted from Rab11a knockdown cells relative to NT-treated cells in glutamine-depleted conditions, following initial normalisation to cell lysate protein. (C') Growth curves are for HCT116 recipient cells pre-treated with EVs isolated as in (C) or with vehicle (PBS). ****P colour denotes significant increase relative to EVs from glutamine-replete cells (black) and Rab11a knockdown cells (red). (D) Left hand group of four images show cells grown in glutamine-replete (2.00 mM) and glutamine-depleted (0.15 mM) conditions for 24 h, then incubated with an anti-CD63 antibody (yellow) for 30 min at 4oC, washed with PBS, chased at 37oC for 30 min, then fixed, immunostained and imaged. Right hand group of four images show cells grown in glutamine-replete and glutamine-depleted conditions for 24 h, incubated with Tfn-Alexa Fluor® 488 (yellow) for 30 min at 4oC, shifted to 37oC for 30 min, then washed, immediately fixed and imaged. (E) Western blot showing levels of anti-CD63 heavy chain and biotin-conjugated Tfn in HCT116 cells cultured for 24 h in glutamine-replete or -depleted conditions, incubated for 30 min in medium containing these molecules at 4oC, then chased at 37oC for 15 min (the chase step was not performed for Tfn in D, to reduce loss due to rapid recycling of Tfn). (F) Western blot analysis of EVs isolated by UC from HCT116 cells in glutamine-replete medium, transduced with a Rab7 or non-targeting (NT) control shRNA knockdown construct. Bar charts show changes in putative exosome proteins in EVs isolated by ultracentrifugation, following normalisation to cell lysate protein. (F') Growth curves are for HCT116 recipient cells pre-treated with EVs isolated as in (F) or with vehicle (PBS).
Figure 4(A) Yeast two-hybrid interaction assays of ASY3 with different ASY1 variants. Monomeric GFP (mGFP) fused with AD (activating domain) and BD (binding domain) were used as controls. Yeast cells harboring both the AD and BD plasmids were grown on synthetic medium supplied with glucose in the absence of Leu and Trp (-L/T, left panel), on Synthetic Drop-out (SD) medium in the absence of Leu, Trp, and His (-L/T/H, middle panel), and on SD medium in the absence of Leu,Trp, His and Ade (-L/T/H/A, right panel). Yeast cells were incubated until OD600 = 1 and then diluted 10-, 100- and 1000-fold for the assays. (B) GST pull down of ASY3 with different ASY1 variants. The numbers above the bands show the relative intensity of the bands. The input and pull down fractions were analyzed by immuno-blotting with the anti-GST (ASY3) and anti-MBP (ASY1) antibodies. (C) Quantification of the pull down fractions of ASY1 as shown in (B). The band intensity in the pull down of ASY11-300/T142V;T184V at a Triton X-100 concentration of 0.5% was defined as 1. The relative amount of ASY1 in the pull down fractions was normalized by the band intensity of the pulled-down ASY3 fraction. The average band intensity of ASY1 at different concentrations of Triton X-100 used was plotted. Asteriks indicate significant difference (two-tailed t-test, p < 0.05). Error bars represent mean ± SD and two biological replicates were performed..