EMBO/sd-character-level-ner huggingface dataset:
Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.
Representative time-lapse image series of the merodiploid recA reporter strain (P2P1recA-GFPdes_RecA-mCherrywt) cultured in fresh (A) or spent (B) flow medium (see Materials and Methods). Fresh 7H9 medium is unlabeled; spent medium (S), ciprofloxacin 1 µg/ml in fresh (CIP) or spent (S-CIP) medium, and SYTOX 0.5 µM (SX) are labeled. Phase-contrast (gray), GFPdes (green), and mCherrywt (magenta) fluorescence are merged. Fluorescence images of each channel are scaled to the brightest frame. Light-green background in the first 4 frames (B) is due to the autofluorescence of spent medium. Cells were imaged at 30-min intervals and numbers represent hours. Scale bar, 10 µm.
Analysis of TFEB binding and modulation of the CDK4 promoter in human ECs. ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as "+IgG") or with Ab anti-TFEB (indicated in the bar graph as "+Ab anti-TFEB"), followed by qPCR for CDK4. Bar graph shows the percent enrichment (n=3, mean±SD).
H) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells compared with DMSO under Ca2+ chelator BAPTA treatment. At least 3 independent experiments were performed. Mann and Whitney test (stv, stv+BAPTA, BAPTA, NSC668394, NSC668394+BAPTA vs DMSO) *p ≤ 0.05.
Quantification of % tumour area (normalised to total lung area) in KPL (n=6) and KPLU (n=5) animals. Quantification of lung tumour numbers in KPL (n=6) and KPLU (n=5) animals.
Figure 8. The multiple substrates of PARN in telomere biology and rRNA biogenesis. PARN regulates the expression of telomere-related gene transcripts, including shelterin transcripts and the hTR RNA component of telomerase. How PARN stabilizes hTR has been well described and involves its 3' end deadenylation. However, at present, the mechanisms underlying PARN-dependent regulation of telomere-related gene transcripts are still unknown and may involve direct or indirect regulations through 3' end deadenylation of either the shelterin mRNAs or some miRNAs that bind to these mRNAs. Our data also show that PARN regulates the level of DKC1 mRNA through the control of p53 levels. Taken together, PARN impacts on both telomere stability and telomere length. An additional role of PARN is to regulate rRNA maturation in both human and mouse cells. Our data suggest that PARN-dependent regulation of rRNA maturation occurs independently of p53. The involvement of PARN in various aspects of cell biology likely explains the severity of the phenotype in Høyeraal-Hreidarsson syndrome patients carrying biallelic PARN mutations.
A Schematic of recombinant Clr4 proteins used in the in vitro HMTase assay: GST-fused full-length Clr4 (FL) and truncated Clr4 proteins (∆2-∆4).
A C99 is first bound at exosites (pink) located in NCT and PEN-2 (stage 1) and then at exosites in the PS1 NTF (stage 2). When bound at these sites in the PS1 NTF, the cleavage domain of C99 gets access to the γ-secretase active site formed by the PS1 NTF/CTF heterodimer for peptide bond cleavage (stage 3).
(c) Dot blot analysis of the lumenal fraction of purified lysosomes from Neuro2a cells co-transfected with tNHTT-60Q-EGFP and R, RS, RHS, RQ or RHQ. PNS, post-nuclear supernatant.
(F) Combined smFISH and immunofluorescence images showing Gadd45b nuclear puncta-presenting neurons were also LAT-positive. Reactivation was induced by treatment with LY294002 and WAY-150138 for 72 hrs. Scale bar, 20 μm.
(C) Comparison of Yki isoforms binding capabilities by dual-luciferase co-IP. Graphs show the normalized co-IP (NcoIP) results for 13 proteins tested against the two Yki isoforms and the negative control (Flag-Firefly).
Ubiquitination analysis of immunoprecipitated TSC1 (F) from activated (anti-CD3 plus anti-CD28 for 2 h) wildtype or Peli1-KO CD8 T cells by immunobloting using anti-ubiquitin Abs detecting total (Ub), K48 (Ub-K48), or K63 (Ub-K63) polyubiquitin chains.
E. A549 cells were treated as described in (D), but following stimulation with 1000 IU/mL of IFN-α2 for 16h were infected with IAV (WSN/33) at an MOI of 0.01 PFU/cell. Viral titers were determined after 24h by plaque assay. Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by 1-way ANOVA on log-transformed plaque counts (***p-value < 0.001). Data information: dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN-α2, respectively. Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions.
B) Relationship between the log2 fold change in RNA level in the nucleus and that in the cytoplasm for all genes upon transfer of AC16 cells from 37°C to different cold temperatures for 24h. Points are color-coded from low to high density (black < blue < red < yellow).
Divergent genes have higher APA variability between replicates compared to conserved genes in each tissue (Mann-Whitney U tests, ***P < 0.001). The boxplot shows the Q1 to Q3 quartile values (the box limits), the median (the horizontal green line) and values within the 1.5 * IQR (the whiskers).
D APP24 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentage of the youngest age group.
(D) SDS-soluble SOD G85R and TDP-43 Q331K species are modified by linear ubiquitin chains. HEK293T cells were co-transfected with disease-associated proteins and either the plasmids indicated or with control siRNA or OTULIN siRNA. 48 h after transfection cells were lysed under denaturing conditions (1% SDS), followed by immunoprecipitation of SOD G85R and TDP-43 Q331K via the HA tag (1% Triton X-100, 0.1% SDS). Immunopurified proteins were detected by Western blotting using the M1 ubiquitin-specific antibody 1E3.
Immunisation with cGAMP-VLPs enhances accumulation of Tfh cells in the draining lymph node. T follicular (Tf) cells were identified by flow cytometry as CD4+CD44+CXCR5hiPD1hi cells and were further subdivided into Tfr cells (FoxP3+) and Tfh cells (FoxP3-). The ratio of Tfh/Tfr is shown in C.
(F-F') A clone of cells in the anterior compartment expressing ci3M following incubation in DiBAC.
Correlation of detected expression between ZEB1 or ZNF596, and LINC00115 from a separate cohort of a total of 75 paraffin-embedded clinical GBM samples. Statistical analysis was performed by Chi-square test.
D) Electron microscopy images of HP1-cTKO cells in the absence of 4OHT (left) and of cells treated with 4OHT for 4 days (right). Boxes represent enlarged images.
(A-F) Cells were either untreated (UT) or treated with neocarzinostatin (NCS) in the presence or absence of PIKK inhibitors. Intensities from 3 independent biological replicates were normalized against reference synthetic standards and are depicted as mean ± SEM.
SUMO conjugation assays performed under multiple turnover conditions as in Fig 4. Experiments were performed in technical triplicates (six times for WT). The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.
B Chitin-induced callose deposition in the MAPKKK56xA plant (Line 1). Seedlings were analyzed at 18 hours after treatment with 10 μM chitin. Data are means ±SD from three independent biological replicates, where each biological replicate consists of two technical replicates. The asterisks indicate statistically significant differences from the WT controls by Student's t-test (P < 0.05).
(E) Two patient-derived KRAS;NF1-mutant LUAD cell lines (PDKN1, PDKN2), as well as the control patient-derived KRAS-mutant/NF1-WT LUAD cell line (PDK), were passaged for 14 population doublings prior to collection. The heatmap displays the most significantly upregulated genes in the PDKN1 and PDKN2 cell lines (relative to the PDK control), with the degree of upregulation depicted by color as indicated in the legend.
(D) IL-8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of stimulus, measured via ELISA.
A. Experimental design for F1 offspring derived from either young or aged fathers (YFO: n=57, AFO: n=39).
J. Interaction between Mfn1 and Mfn2 is not affected by hFis1 overexpression. 293T cells were transfected with empty vector or Myc-hFis1, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG or mouse anti-Mfn1 antibody, followed by immunoblotting with indicated antibodies.
(C) Western blot for PP2A, CLU, Cdc25C-T48, Cdc25C, Wee1 and Cdk1 in PC3 cells transfected with siSCR or siPP2A synchronized and nocodazole released at indicated time points. Vinculin was used as loading control.
B, H1299 cells were transfected with an empty vector (Cont.), a Flag-tagged p53, a USP7-specific shRNA, or the last two plasmids together for 48 h. Western blot analysis was then performed.
Kymographs generated by line-scanning across the diameter of a typical kugel shows that kugel diameter oscillated with a periodicity of minutes, while no such oscillations were observed in adjacent similarly sized cerebral vessels (all images grey LUT; inverted).
A Turbidity and bright-field microscope images of indicated groups (left) and the quantitative analysis of turbidity of samples were measured by absorbance at 600 nm (right). 10 μM cGAS, 5 μM G3BP1, 5 μM BSA and 100 nM dsDNA (60 bp) were used in these assays. n = 3 biological replicates. Data information: Representative images are shown Error bars, mean with s.d. , *P < 0.05, ****P < 0.0001, two-tailed t-test. NS, non-significant. Scale bars, 10 μm
Number of ChgB puncta per neuron in dendrites, axons and the total pool (dendrites + axons). Wilcoxon matched-pairs signed rank test: n.s. = not significant, p = 0.75.
(b) Elevated Hace1 mRNA expression in sTAC mousehearts compared with sham controls, expression levels were normalized to Hprt and Gapdh mRNAs. n=6-9 per group, P=0.004 (unpaired two-tailed Student's t-tests).
A Plants expressing the truncated asDOG1 promoter-deficient version pDOG1shDOG1::LUC are more resistant to drought than plants expressing the full-length genomic version pDOG1::LUC-DOG1. Three independent lines are shown for each construct, following 10 days of water deprivation. Scale bar, 2 cm.
B) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 week old plants of Col-0 and two independent inducible MKKK7AA transgenic lines.
(E) Model of the seven transmembrane G-protein coupled photoreceptor shows the light absorbing molecule, the chromophore retinal (yellow), bound to helix 7 (blue). The red crosses indicate where the Melanopsins in the mutants are truncated: the mutated Opn4.1 has lost all transmembrane helices and the mutated Opn4xb ends after the second transmembrane helix. From the structure-function relationship of the photoreceptor one can deduce that neither of the mutant Melanopsins is functional.
A-E G1-arrested cell cultures were released in YEPD supplemented with CPT (50 μM) (E) Graphical distribution of ssDNA length at the junction (black arrow in (D)) in forks isolated after CPT treatment (40 minutes, 50 μM CPT). Only molecules with detectable ssDNA stretches are included in the analysis. The lines show the median length of ssDNA regions at the fork in the specific set of analyzed molecules. Statistical analysis: Mann-Whitney test; ns, not significant; *, P< 0.05; **, P< 0.01; ****, P< 0.0001. The number of analyzed molecules is in brackets
Here, we present the Connolly (solvent-excluded) surface, with the positions of the H9 and F25 residues highlighted. The accompanying chevron is a short-hand representation of the 6 α-helices of FADD's DED.
A "Ping-Pong" scheme of sterol-regulated SCAP/Insig and SCAP/Cideb interactions. SCAP switches from Insig binding to Cideb binding in the absence of sterol, while the sterol-insensitive SCAP-Y298C consistently interacts with Cideb.
Histograms representing the percentage of KZFPs (identified in Fig. 3B) binding sites falling in TEs (red) or TSSs (blue).
G. Activities of fliCp in Ec-K74A-K87A-C and Ec-K74A-K87A-V strains. L-arabinose was used at final concentration of 0.002%. Data are mean ± SD from three colonies.
Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. b. Representative example of staining on resorted Tregs stimulated with PMA and ionomicin for 4 hours at day 5 (n=4).
Lung SPC+/CD11c+cell numbers of Braf+/LSL−V600E;Npc2+/+ (+/+, n = 3) and Braf+/LSL−V600E;Npc2+/hypo (+/hypo, n = 7) mice at 10 weeks post-AdCre infection.
To determine if ciliary compartmentalisation could be restored, young ts-che-3 mutant adults were transferred to the permissive temperature after being raised at the restrictive temperature. The TZ proteins reassembled at the TZ, with loss of the ectopic accumulations, and IFT protein localisation was also restored-strongest at the basal body, similar to animals grown at the permissive temperature. (c) After 24 hours at the restrictive temperature, TZ proteins appear to begin to accumulate distally within the axoneme in ts-che-3 mutants. This is seen, in small amounts, for NPHP-4 and CEP-290 but with stronger ectopic localisation for MKS-6. The IFT proteins CHE-13 and XBX-1 also show accumulations in the axoneme after 24 hours at the restrictive temperature. Data information: For (A), (B), and (C), fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (TZ markers) or accumulation (IFT markers) for statistical analyses. n = 10 cilia (4-7 animals), Kruskal-Wallis test, *P<0.05, **P<0.01. tz, transition zone; bb, basal body; scale bars are 4 µm
Venn diagram showing overlap of (A) OCT4- regions in sorted EsrrbHi (EH) and EsrrbNeg (EN) E-GFPd1 ESCs.
(B) ARPE-19 cells were nucleofected with the indicated Rag- and TFEB-expressing plasmids. After 12 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting with antibodies against FLAG and GST (used to detect TFEB and Rag proteins, respectively).
A Immunoblot expression analysis of proteins implicated in IFNγ signal transduction in U2OS mock or polyclonal MLLT6 knockout cells treated with or without IFN-γ. Numbers indicate band intensities normalized to GAPDH.
C Subcellular fractionation of SCC cells expressing FAK-WT or interface mutant FAK. Whole cell lysates (WCL) and subcellular fractions were subjected to western blot analysis with anti-FAK, anti-Tubulin (marker for cytoplasmic fraction; C), anti-RCAS (marker for perinuclear fraction; PN) and anti-H4 (marker for nuclear fraction; N). The graph shows densitometric analysis of relative FAK amount of the FAK mutants in the different fractions from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars: s.d. * = p < 0.01, # = p < 0.05. For full blots see source data.
(B) Different constructs fused to the Gal4VP16 domain were used in a γ-secretase reporter assay. Control transfections (pIPspAdApt empty vector and pIPspAdApt-APP without Gal4VP16) show no luciferase activity. APP-Gal4VP16 and NotchΔE-Gal4VP16 activate the luciferase gene upon cleavage and are inhibited by DAPT, L658,458, or compound 32 (125 nM). No luciferase activity was detected using TLNΔE or APP in which the transmembrane domain was replaced by that of TLN (APP/TLNTMR chimaera), indicating that this region is not cleaved by γ-secretase.
, D HeLa cells expressing mCherry-LC3 (magenta) were infected with WT GAS and the indicated gac deletion mutant for 2 h at an MOI of 100 (D) percentages of GcAV‐positive cells were quantified.
A. Immunoprecipitation and Western blot analysis to detect proteins bound to GFP-RAB13 expressed from constructs carrying wt or ∆PMO RAB13 3'UTR. GFP-RAB13 (T22N) expresses a nucleotide-free mutant which binds tightly to putative GEFs. RABIF panel is also shown with adjusted contrast to reveal lower binding to wtGFP-RAB13. B. Quantification of RABIF binding to RAB13 from experiments as in (A). n=6. Bars: mean ± s.e.m..
(D) Enhancers ranked by increasing p65 signals in WT (left) and Brd4 KO macrophages (right).
(A, B) Co-immunoprecipitation of ectopically expressed Myc-CDC20 with ectopically expressed Flag-p65 in HEK293T cells.
H Quantification of the bioluminescence signal emitted by H2030-BrM cells in each organotypic culture with established brain metastases at day 3 normalized by the initial value at day 0 (before the addition of any treatment; DEBIO-0932 was added at 100 nM and chlorpromazine at 20 µM) and normalized to the organotypic cultures treated with DMSO. Values are shown in box-and-whisker plots where every dot represents an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=12-13 organotypic cultures per experimental condition, 3 independent experiments). P value was calculated using two-tailed t-test. I Quantification of the bioluminescence signal emitted by B16/F10-BrM cells in each condition (DEBIO-0932 was added at 100 nM and chlorpromazine at 15 µM) at day 3 normalized by the initial value obtained at day 0 and normalized to the organotypic cultures treated with DMSO. Day 0 is considered 12-16h after the addition of B16/F10-BrM cancer cells and treatment or DMSO. Values are shown in box-and-whisker plots where each dot is an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=30-33 organotypic cultures per experimental condition, 4 independent experiments). P value was calculated using two-tailed t-test.
A LincRNA-EPS and negative control RNA (NC) were transcribed in vitro to label desthiobiotin and pulldown proteins from iBMMs lysate, the differential protein bands from silver-stained SDS-PAGE were cut for mass spectrometry analysis. One of the identified proteins from the band in the red box was PKR.
Distinct tumor cell outcomes in response to CTL attack. Three categories were defined: no hit (no calcium response in the target cell), sublethal hit (transient calcium response and no caspase 3 activity), lethal hit (prolonged calcium response and induction of caspase 3 activity). (F) Time-lapse images (left) of a representative CTL-tumor cell conjugate in each of the three categories are shown together with the corresponding quantification (right) of calcium response and caspase 3 activity over time. T cells are delineated by white dashed lines. Scale bar, 10 µm. Data were pooled from three independent experiments. (**p<0.01, unpaired t‑test).
(A) A scheme of the codon-tag reporter system. A codon to be tested (pink, in this case leucine) and one of the 20 sense codons (gray) are alternately repeated 20 times. This codon-tag sequence was inserted at the end of the sfGFP ORF.
(D) The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that Mitofusin2 (MFN2) underwent rapid degradation within 3 hours of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria-rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 hours 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor ZVAD-FMK (10 μM) was added to cells along with valinomycin to prevent cell death.
Laser-induced CNV experiments demonstrate that treatment with a caspase-1 inhibitor (compared to DMSO treated controls) or a neutralizing TLR2 antibody (compared to isotype matched IgG treated controls) inhibits CNV lesion growth. P-values are shown. N=15 mice/group. Representative CNV lesions in choroidal flat mounts are shown. Scale bars 100 μm.
(B) Lag-times fall reciprocally with the allocation towards required proteins (the fraction of mRNA encoding for required proteins) during the shift. Allocation of translation activity towards different proteins are represented by the weight of red and gray arrows (top).
(d) Box-whisker plots show percentages of inhibition on immune cells chemotaxis by treatment with AMD3100 (CXCR4 small molecule inhibitor). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample sizes are n = 6 NAFLD and n = 4 healthy, with 3-4 technical replicates per donor.
Confocal microscopy reveals robust recruitment of βarr1 to B2R mutant constructs upon agonist-stimulation. HEK-293 cells expressing either B2RWT or B2R mutants along with βarr1-mCherry were stimulated with agonist (Bradykinin; 100nM) and the localization of βarr1 was visualized using confocal microscopy. Representative images from three independent experiments are shown here, and the scale bar is 10μm. Visual scoring from three independent experiments revealed agonist-induced βarr1 recruitment (i.e. membrane and endosomal localization) in approximately 75% of the cells for B2RWT (662 cells), 74% of the cells for B2RΔG368 (169 cells), 75% of the cells for B2RL370T (130 cells), 85% of the cells for B2RΔG368+L370T (132 cells) and 77% of the cells for B2RΔI374 (116 cells).
B HEK-293T cells were transfected with Pim1 and each EDC3 domain (Fig. 2A) fused to the C-terminus of the GFP-tagged vector. The individual fragments were immunoprecipitated with GFP-Trap beads and analyzed by Western blotting with the indicated Abs. EDC3 wild type (W), F1, F2, F3, F4 were EDC3 fragments vector fused to GFP. The C1 lane was only transfected with the GFP Flag vector while C2 contained EDC3 wild type fused to GFP and no Pim1 but empty vector control (pcDNA3).
(H-I) Volcano plot displaying the up- and down-regulation of the proteins altered by Apamin and XE991 (t-test). The blue dots indicate proteins involved in synaptic structure and function.
B-D) PTX3 application induces a remodeling of the PNN in culture, as assessed by the increase mean intensity and integrated density value of the synapse-co-localizing aggrecan signal whereas no difference in the total area of aggrecan is evident (integrated density: Ctr=1±0.103, PTX3=1.874±0.197; mean intensity: Ctr=1.000±0.088, PTX3=1.556±0.107; total area: Ctr=1±0.179, PTX3=0.982±0.149. Number of fields examined: 26 Ctr, 22 PTX3; Mann-Whitney test; 3 independent experiments, data are presented as normalized mean values±SEM)
(D) Lysates from untransfected, CypA-Flag- or Flag-CLIP170-transfected cells were subjected to CA-NC binding assays and WB analysis.
D. Co-immunoprecipitation of FLAG-dmDis3l2 (bait) and GFP-Tailor full-length and truncations expressed in Drosophila S2cells. GFP-Nibbler (Nbr) served as negative control. Domain architecture of GFP-Tailor truncations are indicated.
C, Localization of RH12 in root cells of 5-d-old Arabidopsis seedlings expressing GFP-RH12 under control of the native promoter. The seedlings were grown under 23°C (NS) or incubated at 39°C for 60 min (HS). Scale bars = 10 μm.