EMBO/sd-character-level-ner huggingface dataset:
Serine 687 phosphorylation status determines GFP-Polη function at replication barriers created by Chk1 loss.Figure 4Schematic representation of human Polη showing conserved, putative CDK phosphorylation S/TP sites and cyclin-binding domains (RxL). POL: polymerase domain, UBZ: ubiquitin-binding zinc finger, NLS: nuclear localization signal, PIP: PCNA-binding domain. Representative images of siChk1-depleted U2OS cells showing nuclear localization of GFP-Polη-WT/S687A/S687D. Numbers indicate percentage of cells showing nuclear GFP-Polη (mean+SD, n=2). Scale bar: 100 μm. Western Blot of GFP-Polη in U2OS cells, 48/24 h after transfection with siRNA/GFP(-Polη). Actin was used as loading control. IdU track lengths from U2OS cells transfected with GFP (-) or GFP-Polη-WT/S687A/S687D. >180 fibers obtained from 2 independent experiments were measured for each condition. Number of foci per cell after CSK extraction in siLuc and siChk1-depleted U2OS cells transfected with GFP-Polη-WT/S687A/S687D. >150 cells/sample were analyzed in 4 independent experiments. p<0.05 was considered significant. Representative images of cells with GFP-Polη foci used for quantification in (E). The number of foci per cell for each representative nucleus is indicated. Scale bar: 10 μm. Model that depicts the effect of CDK-dependent phosphorylation of Polη on fork elongation. In Chk1-deficient cells, replication barriers accumulate and recruit Polη. CDKs, whose activity is high in Chk1-deficient cells, phosphorylate Polη, impeding its engagement in fork elongation. Upon CDK inhibition, Polη bypasses replication barriers created by Chk1 loss, promoting fork elongation.
Mcm2‐7 loading is restricted to ORC binding sites in the absence of cyclin EGenome‐wide analysis of ORC localization by ChIP‐chip.Figure 4A Genome‐wide analysis of ORC localization by ChIP‐chip. ORC enrichment from asynchronous cells is depicted for a 5‐Mb section of chromosome 2L.B Genome‐wide analysis of Mcm2‐7 localization in early G1 by ChIP‐chip. Mcm2‐7 enrichment from cyclin E RNAi‐depleted cells is depicted for a 5‐Mb section of chromosome 2L.C Venn diagram depicting the overlap between ORC and Mcm2‐7 peaks.
Figure 5A Expression of BCL-XL (top), BCL-2 (middle) and actin (bottom) in Dusp5+/+ and Dusp5-/- BM-derived eosinophils. Western blot analysis of cytoplasmic fractions isolated from Dusp5+/+ and Dusp5-/- BM-derived eosinophils cultured in the absence or presence of IL-5 (50 ng/ml) or IL-33 (50 ng/ml) for 3 days. Results are representative of four independent experiments. The ratios of BCL-XL and BCL-2 to actin protein levels are quantified at the bottom of each lane.B Quantitation of cytoplasmic fraction associated BCL-XL and BCL-2 protein from Dusp5+/+ and Dusp5-/- BM-derived eosinophils by Western blot analysis from (A). Graph is the relative pixel intensity of cytoplasmic BCL-XL and BCL-2 from four independent experiments including (A). C RT-PCR quantitation of Bcl-xl expression in Dusp5+/+ and Dusp5-/- BM-derived eosinophils treated with IL-33 for 0, 1, 3 or 5 days. Results are representative of three independent experiments. D Cleaved CASPASE 3 levels of IL-5 (20 ng/ml) treated, IL-33 (20 ng/ml) treated or cytokine starved Dusp5+/+ and Dusp5-/- BM-derived eosinophils as determined by Western blot analysis of cytoplasmic fractions. Results are representative of four independent experiments. E CYTOCHROME C (CYT C) levels in cytoplasmic fractions from IL-5 (20 ng/ml) treated, IL-33 (20 ng/ml) treated or cytokine starved Dusp5+/+ and Dusp5-/- BM-derived eosinophils as determined by ELISA. Results are representative of 3 independent experiments.
Figure 2A) Immunopurified C-terminally GFP tagged PRKAG2 N-terminal tail fragments (amino acids 1-136, 1-242) with deletion of Δ122-124 or without (WT) were incubated with full length Ulk1 wild type (WT) or kinase inactive (KI) for in vitro kinase assays. Autoradiograms and Coomassie gels reveal 32P incorporation and protein level respectively. B) GFP-tagged PRKAG2 N-terminal tail fragments (amino acids 1-242) were immunopurified and phosphorylated in vitro by Ulk1 WT or KI. Immunoblot analysis revealed that S124 is phosphorylated by Ulk1 in vitro. C) Full length FLAG-PRKAG2 WT or Δ124 were coexpressed with full length Ulk1 WT and incubated in the presence of 1μM MRT68921. Western blot analysis revealed that Ulk1 overexpression strongly promotes S124 phosphorylation in cells. D) Cells expressing empty vector (EV), or PRKAA1-myc, PRKAB2 and PRKAG2-FLAG (AMPK) alone or with myc-Ulk1 WT (ULK) were cultured in full medium (F), EBSS (St), or serum-free medium (SFM) alone or in the presence of 991 (1µM) or MRT68921 (1μM) for 90 minutes as indicated. 2% Loading control and pull down samples were analysed by Western blot.
Figure 3Representative electron microscopy images of sections of wild type and Ccp1-/-Ccp6-/- cerebral cortices with myelinated axons. In Ccp1-/-Ccp6-/- sections, several myelinated axons show signs of degeneration, such as accumulation of cellular organelles and cytoplasmic densification (arrows), or axonal lysis (stars). Note that only some axons in the observed sections displayed these phenotypes Ccp1-/-Ccp6-/-, while such figures were never observed in wild type cortices. Scale bar: 1 µm.
WT (a), dnm1Δ (b) and atg1Δ cells (c) and atg32Δ cells (d) expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 were incubated in SL medium for 7 days, and 10 OD600 units were sampled at day 1 and day 7 and processed for immunoblotting with anti-GFP antibodies as described in the Methods section.f5WT (a), dnm1Δ (b) and atg1Δ cells (c) and atg32Δ cells (d) expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 were incubated in SL medium for 7 days, and 10 OD600 units were sampled at day 1 and day 7 and processed for immunoblotting with anti-GFP antibodies as described in the Methods section. Positive control used in c and d was from dnm1Δ cells expressing Idp1-GFP from a plasmid at 4-day incubation (Fig. 1b, lane 4). Release of free GFP in WT cells is reproducibly variable between these reporters, and varies between 0 (Idh2) and 60% (Aco2). (e) Densitometric quantification of the release of free GFP (as % of total signal) in four independent experiments, comparing the results between WT and dnm1Δ cells. Bars denote s.d. (n=4).
Kinetochore-microtubule attachment stability is not required for initiating but is required for terminating spindle elongation(A) Live imaging of Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, -9D or -9A.Figure 4(A) Live imaging of Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, -9D or -9A. Z-projection and 3D-reconstructed images show microtubules (EGFP-Map4, green) and chromosomes (H2B-mCherry, magenta). Scale bar, 10 μm. (B) Ndc80-9D is defective for limiting spindle elongation. The aspect ratio (length/width) of 3D-reconstructed spindles was measured over time (n=18, 18 and 18 oocytes, respectively, from 3 independent experiments). (C) Ndc80-9D causes excessive spindle elongation. Spindle lengths measured after 3D reconstruction are shown (n=18, 18 and 18 oocytes, respectively, from 3 independent experiments).
Figure 4(A) Schematic representation of the CAR construct used, consisting of N-terminal anti-hCD70 VHH, hCD8α hinge and transmembrane (TM) domain, CD137 co-stimulatory domain and C-terminal CD3ζ activation domain. (B,C) SKOV3 tumor growth in NSG mice after i.v. injection of 6 x 106 hCD70 CAR T-cells on day 12 and p.l. treatment with 50µg mCD13-AFR (↑). Tumor growth is shown as mean TSI, error bars are SEM (n = 5) (B) or as TSI of individual mice (C). (D) Flow cytometric analysis of hCD45+hCD3+eGFP+ CAR T-cells in SKOV3 tumors on day 18 after tumor inoculation. Mice were injected with 6 x 106 CAR T-cells on day 12 and treated with PBS or CD13-AFR (E) B16Bl6 tumor growth and body weight change after daily p.l. injection of 30µg mCD8-AFN, 50µg mCD13-AFR or a combination thereof. Tumor growth is shown as mean TSI + SEM (n = 6). The line under the graph represents the treatment period. (F) TSI of individual tumors of the indicated treatment groups at day 19 after tumor inoculation.
Figure 3A. Scheme of the assay to determine shRNA strand activities in the absence or presence of specific TuDs. Huh7 cells were co-transfected with the shRNA and the TuD encoded on the same plasmid, and a luciferase reporter with appropriate shRNA strand binding sites. bs, binding site. B. Measurement of resulting luciferase activities in cell lysates 48 h post-transfection. Note the potent inhibition of shHBV7 sense strand activity with the specific TuDHBV7, without interference with the antisense strand (compare left two orange or blue bars, respectively). bs, binding site. C. In vivo evaluation of different shRNA and TuD combinations after packaging into double-stranded AAV8 vectors and intravenous (i.v.) injection of 1x1011 particles into HBV-transgenic mice (HBV1.3.32). Anti-HBV activity was determined by measuring HBsAg and HBeAg levels in the serum of treated animals at the indicated time points post-AAV injection.
Figure 5A. Representative normalised DRIP-seq data in two genes COL22A1, spanning over 200 kb, and MYC. The locations of H-DNA and G4 motifs are shown below the gene map. Wild type in blue; primpol in red. The corresponding RNAse H-treated samples are dashed. See Methods for further details of graphic generation. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR). B. Metagene analysis of DRIP peak distribution in wild type and primpol DT40 cells compared with the distribution of the indicated features in the genome. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR). C. DRIP peak heights in wild type and primpol DT40 normalised to Drosophila S2 spike-in. n (wild type) = 41445; n (primpol) = 48648. D. Correlation of normalised DRIP peak heights in the overlapping peaks between wild type and primpol. Blue line = 1:1 correlation; red line = linear regression through data. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR). E. Correlation between H-DNA forming sequences and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). F. Normalised DRIP peak heights in the genes identified as associating with H-DNA. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR). G. Correlation between G4 motifs ([G3-5N1-7]4) and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). H. Normalised DRIP peak heights in the genes identified as associating with G4 motifs ([G3-5N1-7]4). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).
RAB21 is required for endosomal sorting of specific cargo types.Figure 6(A-C) RAB21 is not required for CI-MPR or GLUT1 trafficking. (A) Immunofluorescence of endogenous CI-MPR and TGN46 or GLUT1 with LAMP1 in RAB21-knockout cells. Boxed region is magnified. (B) Per cell Pearson Correlation between CI-MPR and TGN46 or (C) GLUT1 and LAMP1; SEM, n=3 independent experiments. Data information: In (A), scale bars represent 20µm or 5µm in enlarged views for CI-MPR, while they represent 10µm in enlarged views for GLUT1. Statistical tests used: (B) - Unpaired t-tests (C Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats MCT1 trafficking requires RAB21. (D) Immunofluorescence of endogenous MCT1 in wild type or RAB21-depleted cells. (E) Per cell integrated MCT1 intensity (in Relative Fluorescence Unit); SEM, n=2 independent experiments. Data information: scale bars represent 20µm or 5µm in enlarged views. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats (F) RAB21 interacts with SLC3A2. FLAG immunoprecipitation of FLAG:RAB21 and immunoblot of co-expressed SLC3A2:HA. Lysates correspond to 5% input, n=3 independent experiments. (G-H) RAB21 is required for appropriate SLC3A2, Basigin and CD44 trafficking. (G) Antibody-uptake assays in parental or knockout RAB21 cells. Cells were stained with an Alexa488 conjugated anti-mouse antibody. Arrowheads point to endosomal tubules. Note that non-internalized antibodies were removed by an acid wash, which was not fully efficient for CD147 and CD44, explaining plasma membrane labeling. (H) Percentage of cells with tubules in parental or RAB21 knockout cells; SEM, n=3 independent experiments. Data information scale bars represent 20µm or 5µm in enlarged views. Statistical tests used Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats
Figure 1(A) A schematic representation of targeted deletion of mouse Brd4. The exon-intron organization of the Brd4 locus (top). Targeting vector contained exon 3 flanked by LoxP sites (green) and a neomycin resistance gene flanked by FRT (white). Cre mediated recombination should cause deletion of exon 3 resulting in loss of BRD4 expression (bottom). (B) Genotyping of Brd4fl/fl mice with ERT2-Cre (Cre) or without ERT2 -Cre (+/+). Tamoxifen treatment deleted 2.2 kb Brd4fl/fl fragment from Cre/+ and Cre/Cre mice (top). Cre/+, Cre/Cre and +/+ mice were confirmed by the presence of ERT2 -Cre band (Cre), or its absence (WT) (bottom). (C) WT and Brd4 KO cells were tested for mRNA (left) and the protein (right) by qRT-PCR and immune blot assays. Values represent the average of three experiments +/- SD. (D) Vav-cre mediated Brd4 depletion was verified for cells from Brd4fl/fl Vav-Cre mice (fetal liver or spleen) by qRT-PCR and Immunoblot (left and right). Values represent the average of three experiments +/- SD. (E) Photographic images of E14.5 embryos from WT and KO mice. (F) Corresponding Flow cytometric profile of Lin- fetal liver cells stained with C-Kit and Sca-1 antibodies examining early hematopoietic progenitor population (LSK) (left). LSK populations were further divided by the Flt3 marker to detect HSCs and MPPs (middle). The number of total cells, that of LSKs, HSCs, MPPs in a fetal liver were estimated on indicated days of embryonic development (E13.5, E14.5 and E17.5). Values are the average of 3-5 embryos +/- S.D (right). (G) A schematic of hematopoietic stem cell and early progenitor cell pathways. (H) Representative flow cytometric profile of CD71- cells stained for the myeloid and lymphoid markers. Values are the average of 3 E17.5 embryos +/- S.D.
LAT is not required for CAR microcluster formation.Figure 3A. WT or LAT-deficient Jurkat cells (Jcam2.5) expressing CAR-GFP were stimulated on SLB-coated with OKT3 (anti-TCR antibody) or CD19 (CAR antigen); in both conditions, the lipid bilayers were also coated with ICAM-1 to facilitate cell attachment. TIRF microscopy revealed clustering of Ax647-labeled streptavidin-Biotin-OKT3 or CD19, which serves as a probe for TCR or CAR respectively. Scale bar, 2 µm. B. Quantification of clustering as normalized variance. n = 100 cells. Shown are the mean ± SD. Statistical test: unpaired two-tailed t-test. **** p < 0.0001. ** 0.005 < p < 0.05.
Figure 3A) Correlations of mean fold-changes of the proteins changing PD-dependently in the Columbia and LCC cohorts. Only proteins quantified in both cohorts are shown (n=330). Proteins overlapping between the two cohorts are labeled with their name. B) Fisher exact test to identify significantly enriched GO-terms in the PD-associated proteins in urine. Importantly, the enrichment score of the Fisher exact test does not indicate if the proteins were up- or downregulated in PD patients but rather that the regulated proteins - independent of the directionality - compared to the total urinary proteome are associated with the enriched term. All GO-terms that were significant in both cohorts are displayed (FDR < 5%
Figure 1A. Physical map of the locus of TRIM46 [chromosome 1: 155173381-155184971 (hg38)] and SNP rs4971059 [chromosome 1: 155176305 (hg38)]. B. HaploReg v4.1 was used to predict the potential functions of SNP rs4971059 (left). The yellow box means "Weak Enhancer 2" (H3K4me1 and DNase enrichment), the orange box means "H3K4me1 Enhancer", and the black box means "missing data". ChIP assays were performed with antibodies against H3K4me1, H3K4me3, or H3K27Ac in the region surrounding SNP rs4971059 (right). C. Luciferase assays showing enhancer activity in the SNP rs4971059 region in MCF-7 cells. MCF-7 cells were transfected with the indicated promoters: PGL3-SV40, PGL3-SV40-SNP[G] or PGL3-SV40-SNP[A] (left); PGL3-TRIM46, PGL3-TRIM46-SNP[G] or PGL3-TRIM46-SNP[A] (right). Forty-eight hours after the transfection, luciferase activity was measured. Relative luciferase activity was calculated as firefly luciferase activity divided by renilla luciferase activity and shown relative to the control. D. The SNP rs4971059-containing region formed close contact with the promoter of TRIM46 in a 3C assay. DpnII fragments in 1q22 were shown on the top panel (hg38). SNP rs4971059 resides in 3C fragment 17, and fragment 5 is in the genomic region 3-kb upstream of the TSS of TRIM46. PCR primers were designed near the end of DpnII fragments in the same orientation and 3C assays were performed with a constant primer in fragment 17 (lower-left panel) or a constant primer in fragment 5 (lower-right panel). E. eQTL analysis demonstrating the correlation between rs4971059 genotype and expression of TRIM46. The panel showed results from breast cancer tissues obtained from the mastectomy of the patients. In the boxplot, the central lines, the box limits, and the whiskers represented medians, 25th/75th percentile, and min/max, respectively (FDR=0.00147). F. A physical 650-kb window of the genomic distribution of genes surrounding SNP rs4971059, such as EFNA4, KRTCAP2, MUC1, MIR92B, THBS3,and DAP3 (top). Total RNAs were extracted from [GG], [GA], or [AA] MCF-10A cells and analyzed for the expression of EFNA4, KRTCAP2, TRIM46, MUC1, THBS3, MIR92B, or DAP3 by RT-qPCR. TRIM28 was examined as a negative control (bottom).
a) Representative images of Zellweger cells (GM13267) showing endogenous TSC2, TSC1 and Rheb (green) co-localization with PMP70 (red).figf5(a) Representative images of Zellweger cells (GM13267) showing endogenous TSC2, TSC1 and Rheb (green) co-localization with PMP70 (red). Scale bars, 15 μm. (b) Western analysis of human fibroblasts obtained from a Zellweger (GM13267) or a corresponding control patient (with Ehlers-Danlos syndrome (GM15871)) treated with the indicated doses of H2O2 for 1 h. mTORC1 signalling was assessed by western analysis for pS6K (T389), S6K, pS6 (S235/236), S6, p4E-BP1 (T37/46), 4E-BP1, pATM (S1981), ATM, pAMPK (T172), AMPK, p62 and LC3. (c) Western analysis of human fibroblast (GM13427) cells pre-incubated with 3 mM N-acetyl cysteine (NAC; ROS scavenger) for 1 h before being treated with 0.4 mM H2O2 for 1 h using anti-pS6K (T389), S6K, p62 and LC3 antibodies. (d) Representative western analysis using cell extracts from human fibroblasts obtained from a Zellweger patient (GM13267) or control fibroblasts (GM15871) treated with amino-acid-free media for 60 min, and stimulated with a mixture of amino acids for 10 min. mTOR signalling was monitored using anti-pS6K (T389), S6K, pS6 (S235/236), S6, p4E-BP1(T37/46) and 4E-BP1 antibodies. Uncropped images of western blots are shown in Supplementary Fig. S6.
Figure 5(A) Image of touch neurons viewed by zdIs5 in different genetic backgrounds. Scale bar: 20 μm. (B) Quantification of the percentage of animals with 0, 1, 2, 3, 4, 5 and 6 surviving touch neurons in different genetic backgrounds. Data show mean ± SEM. At least 50 animals per strain were analyzed in each experiment. Number of experiments n = 5. (C) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds in (B). The lid-1(xd288) and atgl-1(xd314) mutations significantly increase the number of surviving neurons. Neuron-specific expression of atgl-1 or lid-1 reverses the protective effect of atgl-1 or lid-1 mutation. The data were analyzed using one-way ANOVA with Turkey's multiple comparison test. Asterisks signify significant differences between the groups under the crossbars. ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n = 5, with at least 50 animals per strain analyzed in each experiment. (D) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds. Mutations of fat-1, fat-3 and fat-4 significantly enhance the suppression effect of atgl-1(xd314) in preventing the neurodegeneration of mec-4(d). The data were analyzed using one-way ANOVA with Bonferroni's multiple comparison test. ### and #### denote significant differences as compared to atgl-1(xd314); mec-4(d). Asterisks signify significant differences as compared to mec-4(d). ns: not statistically significant. ****P < 0.0001. ###P < 0.001, ###P < 0.0001. Data show mean ± SEM. Number of experiments n ≥ 3, with at least 50 animals per strain analyzed in each experiment. (E) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds supplemented with different fatty acids. AA and EPA but not LA significantly enhance the neurodegeneration triggered by mec-4(d). The data were analyzed using one-way ANOVA with Bonferroni's multiple comparison test. Asterisks signify significant differences between the groups under the crossbars. ns: not statistically significant. ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n ≥ 3, with at least 50 animals per strain analyzed in each experiment.
Figure 7A - Frequency of rosette formation in the experimental conditions indicated. n, number of embryos per condition. B-G - Distribution of neurons (Tuj1, red), GFP (green) and mitoses (PH3, white) in the rostral telencephalon of wild-type E14.5 mouse embryos electroporated at E12.5 with the indicated plasmid combinations. Inset shows the same field of view, with location and extent of electroporations. In (C) and (E), arrows indicate rosettes and arrowheads indicate neuronal ventricular ectopias. (D) and (F) are high magnification details of individual rosettes (dashed lines), where arrowheads indicate apical and basal mitoses. Loss of endogenous let-7 drives the formation of rosettes and ventricular neuronal ectopias (C), which is rescued with the additional loss of Irs2 (G).
Figure 5A, Western blot of DNA damage signalling in BRPF3 depleted cells. siRNA transfected U-2-OS cells were treated with HU for 2 h or 24 h. One representative experiment out of two biological experiments is shown. B-C, Recovery from replication stress. siRNA treated cells were treated for 24 hrs with HU (0 h), and released into normal medium for the indicated times (see also Fig. S6A, B). (B) DNA replication rate measured by EdU incorporation. Cells were pulsed with EdU for 15 min and gated as shown in Figure S6A to analyse EdU positive cells. n > 1000. Mann-Whitney: **** P < 10-4. (C) Cell cycle progression measured by FACS analysis of DNA content. One representative experiment out of two independent biological replicas is shown.