EMBO/sd-character-level-ner huggingface dataset:
Figure 6A, B Step-wise assembly of MAC on convertase-labeled bacteria. Convertase-labeled bacteria were incubated with C5/C6 or C5/C6/C7 for 15 minutes, and subsequently washed (@) or treated with 10 μg/ml Eculizumab (Ecu). Then, the remaining MAC components (C7-9 for C5/C6 or C8-9 for C5/C6/C7 respectively) were added to the incubation mixture. In the control conditions (Conv-MAC), the remaining MAC components were added to the incubation mixture without washing or adding an inhibitor. (A) Outer membrane (mCherry) and (B) inner membrane damage (% Sytox positive) were determined. Data information: (A-B) Data represent mean ± SD of 3 independent experiments. Statistical analysis was done using a ratio paired two-tailed t-test in which the test conditions were compared to Conv-MAC and displayed only when significant as * p≤0.05.
Figure 5(A) qRT-PCR analysis of the mRNA levels of the genes related to β-catenin downstream signaling pathway. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments. (B) qRT-PCR analysis of the mRNA levels of NFκB targeting genes. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments. (C) Immunoblotting analysis of 22Rv1-P cells with different ESM1 overexpression in the nucleus and cytoplasm were performed with the antibodies indicated. (D) Suppression of β-catenin or NFκB in 22Rv1-P cells stably expressing ESM1-NLS were treated with docetaxel at the indicated concentrations for 48 h. ** P < 0.01 when compared to vector-shScramble cells by two-tailed Student's t test and error bars represent the standard deviation of three independent experiments. (E) Tumor spheres formed by 22Rv1-P cells stably expressing ESM1-NLS with either β-catenin or NFκB shRNAs. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ## P< 0.01 when compared to vector-shScramble cells, ** P< 0.01 when compared to ESM1-NLS-shScramble cells by two-tailed Student's t test. (F) Representative images and quantitative data comparing cell invasion of the indicated cells. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ## P< 0.01 when compared to vector-shScramble cells, ** P< 0.01 when compared to ESM1-NLS-shScramble cells by two-tailed Student's t test.
Figure 5Representative images of mESC neuronal differentiation as in Fig 4B. Shown are XRCC1wt and XRCC1null (XRCC1null1, XRCC1null2) mESCs in 2i medium supplemented with LIF (2i LIF) and in ESC medium supplemented with LIF (ESCM LIF), EBs in ESCM LIF and 5µM RA (EB 4d ESCM RA) and dissociated neuron-like cells in B27 (B27). Scale bars 25µM, 50µM, 500µM as indicated. Neurite grids were observed for XRCC1wt; heterogeneous cell populations formed with the XRCC1null (XRCC1null1, null2) clones. Quantification (below) of neural cells with neurites of total differentiating XRCC1wt, XRCC1null mESCs. n = 5. Means with standard deviation (SD). **: P<0.01. Non parametric, Mann-Whitney test. Quantification of mRNA expression of pluripotency and ectodermal/neurogenesis marker genes by RT-qPCR. n ≥ 3. Mean values and standard deviations (SD) of EBs relative to their mESC (ESCM LIF, 16h), normalised to Gapdh. SUMO coordinates BER in active DNA demethylation. SUMOylated XRCC1/BER-proteins are recruited to DNA-bound TDG to form the BERosome (grey oval) SUMO protease (SENP2) liberates SUMO from XRCC1 for attachment to TDG by the SUMO-conjugating enzymes SAE1/2 and UBC9. SUMOylated TDG dissociates from the DNA AP-site and the BERosome, facilitating completion of demethylation by BER, and neuronal differentiation.
Figure 2A. IHC in DRG sections for pHDAC3 revealed that HDAC3 phosphorylation is decreased, primarily in the nucleus 24h after sciatic nerve axotomy (SNA) but not spinal dorsal column axotomy (DCA). Scale bars, 100 μm. B-C. Bar graphs show mean fold change levels (B) or % of pHDAC3+ cells (C) (pHDAC3+ threshold was set at the minimum intensity level of sham cells) ± s.e.m. DAPI counter staining was used to label nuclei N= 9-10 biological replicates. (***p<0.005) indicate significant difference versus sham or laminectomy (lam) (ANOVA followed by Bonferroni test). D. Western blot revealed that HDAC3 phosphorylation is decreased in DRG nuclear extracts but not in the cytoplasm 24h after SNA. E. Bar graphs show intensity measurement of nuclear vs cytoplasm immunoblot. Data is expressed as mean ± s.e.m. N= 3 biological replicates. (***p<0.005) indicate significant difference versus sham (Student´s t-test). F. HDAC3 activity (HDAC3 IP and activity assay) is strongly reduced after SNA but not DCA. Data is expressed as mean ± s.e.m. N= 3 biological replicates. (*p<0.05) indicate significant difference versus sham (ANOVA followed by Bonferroni test).
Figure 8Upon virus infection, RIG-I binds to viral dsRNA and activates MAVS with the help of Riplet. Furthermore, MAVS recruits TRAF3 through TRAF3IP3 to activate downstream TKB1-IRF3 and MAVS activates IKK-NF-κB probably by directly binding to other TRAFs. IRF3 and NF-κB entire the nucleus to turn on type I IFN expression.
Parkin exerts E3 activity only when the mitochondrial membrane potential is decreased.Figure 2.(A) HeLa cells expressing wild-type Parkin or E3-inactivating mutations were treated with CCCP and then immunostained with the indicated antibodies. When E3-inactivating mutations were introduced into Parkin, the mitochondrial ubiquitylation signal disappeared. (B and C) HeLa cells expressing HA-Parkin (B) or expressing both Mt-GFP and HA-Parkin (C) were treated with CCCP or DMSO (control) and then immunostained with the indicated antibodies. (D) Localization of GFP-Parkin to the mitochondria after CCCP treatment. (A-D) Higher magnification views of the boxed areas are shown in the insets. (E) HeLa cell lysates expressing GFP-Parkin were immunoprecipitated by anti-GFP antibody, followed by immunoblotting with the indicated antibodies. (F) Straight immunoblotting of HA- and GFP-Parkin in the absence or presence of CCCP. Note the slower migrating ladders derived from ubiquitylation (Ub) in only the GFP-Parkin with CCCP lane. (G) GFP-Parkin-expressing HeLa cells with various pathogenic mutations (Fig. 1 C) were treated with CCCP and subjected to immunoblotting. Asterisks show ubiquitylation of GFP-Parkin. Vertical black lines indicate that intervening lanes have been spliced out. IB, immunoblot; IP, immunoprecipitation.
Figure 4A-C CRISPRa activation of ADAM22 (a), ABCB1 (b) and PTN (c) in human RPE-1 cells. Top panels visualize DamID data similar to Figure 2a-b. Middle panels show maps of replication timing at the same resolution and in the same plotting style as panels, except that different colors are used as indicated. Bottom panels show gene track, with activated gene highlighted in green.
Tumor-specific CCR9 impedes Th1-type immune response.Figure 5A, B ELISpot assay showing IFN-γ (A) and granzyme B (B) secretion by survivin-specific T cells, as spot numbers, upon CCR9 knockdown (black bars) in MCF7 cells compared to the control knockdown (white bars). T cells (TC) alone (grey bars) were used as control for background spot numbers. C Luminex assay showing cytokine levels in the supernatant from the coculture of survivin-specific TC and either CCR9hi MCF7 (transfected with CCR9-specific siRNA) or CCR9lo MCF7 (transfected with control siRNA) cells. D Phospho-plex analysis showing the phospho-STAT levels in survivin-specific TC upon encountering CCR9hi or CCR9lo MCF7 cells. Log2 ratio of mean fluorescent intensity (MFI) of the respective analytes to the unstimulated TC is plotted herein. E Immunoblot analysis showing the phospho-STAT1 levels in the CCR9hi-treated, CCR9lo-treated or unstimulated TC using the phospho-specific STAT1 (pTyr701) antibody. Beta-actin was used as the loading control.
Figure 5A. Real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D, followed by infection with VSV (MOI, 0.1). B. Immunoblot analysis of p- TBK1, -IRF3, -P38 of cells as in (A). C. FACS analysis of RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D infected with VSV-eGFP at MOI of 1. D. Immunoblot analysis of phosphorylated-TBK1, -IRF3, -P38 in lysates of RKIP+/+ or RKIP-/- bone marrow derived macrophages (BMDM) transfected with flag-RKIP, flag-S109A and flag-S109D, and then stimulated with VSV (MOI, 0.1). E. ELISA of IFN-β and TNF-α of cells in (D).
Figure 4C57BL/6J male mice fed on a high-fat diet drank water with or without asparagine (1.5g L-1) for 18 weeks. In the last 4 weeks, mice were injected CL316,243 daily (0.5mg kg-1 body weight). Body weight of mice in (A) during the high-fat diet feeding. n=5 mice each group. Glucose tolerance test was performed after a high-fat diet feeding for 17 weeks in mice of (A). n=5 mice each group.   Serum insulin levels in mice of (A). n=5 mice each group.   Serum triglyceride levels in mice of (A). n=5 mice each group.   Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test. Fat mass weight in mice of (A). n=5 mice each group. Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test. Representative histology pictures for BAT, inguinal WAT (iWAT) and epididymal WAT (eWAT) in mice of (A). BAT and inguinal WAT (iWAT) UCP1 and PPARγ protein levels in mice of (A).
Figure 1Immunoblot analyses of TDG and TDG-SUMO in whole cell extracts of mESCs and MEFs. TDG and TDG-SUMO conjugates before (2i, 0 h) and during differentiation of TDG wildtype (TDGwt) and TDG deficient (TDGnull) mESC and MEFs (5 µM all-trans retinoic acid (RA, 24 h, 48 h). Percentages of TDG-SUMO relative to total TDG signal are indicated. Immunoblot analyses of TDG, TDG-SUMO in whole cell extracts of mESCs TDG and TDG-SUMO before and after H2O2 treatment (10 mM, 15 min). Immunoblot analyses of TDG and TDG-SUMO in whole cell extracts of mESCs Chromatin fractionation from mESCs expressing wildtype (ESC-TDGwt) or SUMOylation-deficient TDG (ESC-TDGsnm). Sol, chromatin unbound proteins; Chr1, chromatin associated proteins; Chr2, chromatin bound fraction; Histone H2B, stably chromatin bound marker. Immunoblot analyses of XRCC1 and , XRCC1-SUMO, in whole cell extracts of mESCs and MEFs. XRCC1 and XRCC1-SUMO conjugates before (2i, 0 h) and during RA-induced differentiation (24 h, 48 h) in mESCs and MEFs. Percentages of XRCC1-SUMO relative to total XRCC1 signal are indicated. Immunoblot analyses of XRCC1 and XRCC1-SUMO, in whole cell extracts of mESCs XRCC1 SUMOylation in native whole cells extracts before and after incubation with purified SUMO protease (SENP2364-489). Immunoblot analyses of , XRCC1 and XRCC1-SUMO in whole cell extracts of mESCs XRCC1 SUMOylation before and after exposure to H2O2 (5 mM, 15 min). Immunoblot analyses of TET1 isoforms in whole cell extracts of mESCs and MEFs. TET1 and TET1s isoform protein levels in mESCs during RA induced differentiation and in MEFs. Fluorescence microscopy of GFP-TDGwt GFP-TDGpip in mESCs. Scale bar: 100µM. Quantitation of fluorescence signals of GFP-TDGwt and GFP-TDGpip in mESCs. median with interquartile range. >470 cells were analysed of each GFPTDGwt and GFPTDGpip ESCs. Immunoblot analysis of XRCC1 and XRCC1-SUMO in GFP-TDGwt and GFP-TDGpip mESCs.
Figure 8A. KT299 (MATa hkr1Δ msb2Δ ssk2/22Δ) was exposed to the indicated concentrations of α-factor for 15 min in the absence of osmostress. Phosphorylation of Fus3 and Kss1 was detected by immunoblotting. B. KT299 (MATa ssk2/22Δ hkr1Δ msb2Δ) was exposed to the indicated concentrations of α-factor for 15 min in the presence or absence of 0.8 M NaCl, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Data information: (B) Representative results from three independent experiments. C. Average values of four independent experiments were plotted. Data information: Error bars are SEM (C) n=4 D. HM06-1 (MATa ΔS/O/H/M ssk2/22Δ) was exposed to the indicated concentrations (log scale) of α-factor for 15 min in the presence or absence of 1.0 M NaCl, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Average values of three or more independent experiments were plotted. Data information: Error bars are SEM: (D), n=3 or more E. KT306 (MATa hkr1Δ msb2Δ ssk2/22Δ hog1Δ) was transformed with pRS416-Hog1 (WT) or pRS416-Hog1-N149H D162G (N/H D/G), and was exposed to 10 μM α-factor for the indicated time in the absence of osmostress, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Average of three independent experiments are plotted. Data information: Error bars are SEM (E) n=3. F. A schematic model showing that the lack of osmotic enhancement of the Pbs2-Hog1 reaction prevents the pheromone-to-Hog1 crosstalk. G. Typical FRET (YFP/CFP ratio) images showing p38 activation. HeLa cells carrying the p38 reporter PerKy-p38 (Tomida et al., 2015) were stably transfected with an expression vector for the indicated p38α mutant proteins. FRET analysis was performed as described in Materials and Methods. H. Distribution of p38 activity in individual cells from sets (a) - (d) in (G). Data information: (G) Scale bars: 20 μm. (H) Statistics, Student's two-tailed t-test.
Figure 3EVs were purified by SEC from supernatants of Jurkat cells. (A) EVs were subjected to immuno-isolation with beads coupled to antibodies against CD81 or CD63. Bead-associated (Pull-down: PD) vesicles and those left behind (Flow-Through: FT) were loaded on a gel for Western blot analysis with antibodies specific for CD63, Syntenin-1, CD81, ADAM10 and CD3G. A representative blot (top panel) and quantification (mean ± SD) of the proportion of signal in FT as compared with total (PD+FT) from four independent experiments (bottom panel) are shown. (B) Representative images of double-immunogold labelling of EVs purified by SEC from Jurkat cells. Left panel, CD81/CD63; right panel, CD81/CD3E. Arrows show CD63 staining in CD81+CD63+ EVs. EVs were purified by SEC from supernatants of Jurkat cells. (C,D) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with 2 x 109 particles. Captured EVs were detected with either APC-labeled anti-CD81, anti-CD63 or anti-CD3E. (C) Median APC fluorescence values for the different bead populations is shown as the ratio to the median APC fluorescence of control beads (log10 scale). shown. (D) Heat-map representation of the median APC fluorescence values for the different bead populations detected with anti-CD63 or anti-CD3E antibodies relative to the values detected with anti-CD81.
Figure 5A-C. Human patient samples demonstrate relative differences in the immunoexpression of AR, SDHA, p-CaMKK2/p-AMPK, p-p38, and p-Hsp27 in untreated (n=70), neoadjuvant-treated (NHT) (n=130) and CRPC (n=24) samples. Representative images (A), dot plots (B), compiled bar graph (C) support the AR-SDH loop where inhibition of AR axis in NHT samples reduced SDHA levels along with upregulation of p-CaMKK2/p-AMPK/p-p38/p-Hsp27 axis. AR-positive CRPC samples restored SDHA as well as the whole axis. Data information: Data shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. **, p<0.01; ***, p<0.001 and ****, p<0.0001. Scale bar: 100µm. All the TMAs were constructed from minimum 2 cores per patient.
Ectopic human S100A9 expression in CD4+ T lymphoid cell lines potently restricts X4-tropic wild-type HIV-1 infection)(A) E2C- and S100A9-expressing CEM and (D) E2C- and S100A9-expressing Jurkat CD4+ T cells were analysed for the expression of E2C and S100A9 by flow cytometry .Figure 7E2C- and S100A9-expressing CEM and E2C- and S100A9-expressing Jurkat CD4+ T cells were analysed for the expression of E2C and S100A9 by flow cytometry . Cells from above were infected with X4-tropic HIV-1 for 72h and processed for flow cytometry analysis upon staining with anti-HIV Gag p24-FITC. Dot-plots from representative experiments of (B) CEM or (E) Jurkat infected with X4-tropic HIV-1 (50ng of p24 per 105 cells) are shown. Control cells (NI) correspond to non-infected E2C-expressing cells. The percentage of HIV-Gag p24+ cells from four independent experiments for CEM (n=4) and two for Jurkat (n=2) analysed by flow cytometry were graphically represented as means of HIV-Gag+ cells ± SD. When applicable, student t-test was performed to determine statistical significance (**P<0.01;***P<0.001).
Figure 1We included 126 studies in our curated cancer metabolomics dataset, studies that described profiling in multiple cancer types were counted once for each type, giving rise to 136 "cancer vs control" cohorts spanning 18 different cancer types, 71 assessing blood, 39 tissue and 26 urine. We also included 18 diabetes mellitus type 2 studies to determine if our vote-counting method could identify distinct metabolic signatures in cancer versus diabetes.
ULK1 is upregulated and translocates to fragmented mitochondria in response to mitophagy induced by hypoxia or FCCPLevels of ULK1 in MEFs exposed to hypoxia (1% O2) for the indicated times.Figure 1A Levels of ULK1 in MEFs exposed to hypoxia (1% O2) for the indicated times.actin, Western blot B Quantification of the ratio of ULK1 to actin in (A). C Levels of ULK1 in MEFs treated with FCCP (20 μM) for the indicated times.actin D Quantification of the ratio of ULK1 to actin in (C). E MEFs were exposed to hypoxia (1% O2) for 12 h or FCCP (20 μM) for 6 h and then fixed by 4% paraformaldehyde. Cells were stained with anti‐TIM23 (mouse) and anti‐ULK1 (rabbit) primary antibodies, then Alexa Fluor‐488‐labeled donkey anti‐rabbit IgG and Alexa Fluor‐555‐labeled donkey anti‐mouse IgG secondary antibodies before analysis by immunofluorescence microscopy. Scale bar, 10 μm. F Immunoblots of subcellular fractions from control MEFs or MEFs that were exposed to hypoxia (1% O2) for 12 h. PNS, post‐nuclear supernatant; Cyto, cytosol; Mito, mitochondria.ULK1 G Immunoblots of subcellular fractions from control MEFs or MEFs that were treated with FCCP (20 μM) for 6 h. PNS, post‐nuclear supernatant; Cyto, cytosol; Mito, mitochondria.ULK1
A genome-wide RNAi screen designed to identify novel NMD factors in C.Figure 1The PTCx NMD reporter consists of a fusion between GFP (in green) and LacZ (in blue) genes; a PTC is present downstream of the GFP coding region, making the transcript an NMD substrate.
Figure 7Figure 7. Model of RING dimerization in different TRIM ligases.TRIM25 and TRIM32 use different mechanisms to allow their respective RING domains to dimerize: In TRIM25 the propensity to dimerize is low and happens in an intramolecular fashion possibly aided by E2~Ub and substrate binding. It is possible that the B-box domains may contact the CC region to support dimerization. The RINGs of TRIM32 are constitutive dimers even in the absence of the rest of the remainder of the protein or additional binding partners. Tetramerization of TRIM32 may be important for the recognition of low affinity substrates or may allow co-localization of different binding partners.
The Beclin1-binding α1 Helix of vBcl-2 is Required for Inhibition of Autophagy during γHV68 Infection.Figure 4(top) Representative confocal images of GFP-LC3 and HA-tagged vBcl-2 (WT and mutants) in NIH3T3 cells infected with the indicated viruses (MOI = 5). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). Scale bar, 5 µm. (bottom) Quantification of the percentage of virally infected cells with GFP-LC3 punctate staining. Results shown represent mean±SEM of combined results from three independent experiments (200 cells per experimental condition). *, P<0.01 versus HA-WT; **, P<0.001 versus HA-WT.
Figure 7a, Time-lapse images of RPE1 WT and SEPT2-KO cells stably expressing ARL13B-GFP and γ-tubulin-mRuby2. Cells are starved for 32h before the beginning of inspection and imaged every 15min for 9.5h. Scale bar: 5µm. b, Quantification of (a) showing changes in cilia length during time-lapse imaging. Three biological replicates, 10 cilia per sample and repetition. c, Time-lapse images show ectocytosis events observed in RPE1 SEPT2-KO cells. Experiments are done as in (a) with exception that cells are imaged every 15min. Scale bar: 2.5µm. d, Percentage of cilia that remained intact, underwent ectocytosis during live-cell imaging from (a) and (c). Three 3 biological replicates, n>100 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test. e-f, Determination of the cilia breakage point during live-cell imaging of RPE1 SEPT2-KO ciliated cells. The ectocytosis point was calculated by dividing the cilia length at the time of breakage (b, t0) by the length of the cilium one time-point prior to t0 (a, t-1), as depicted in (e). The graph in (f) shows that breaking points (b/a) higher than 0.5 (i.e. breakage towards the cilia tip) are more frequent. Three biological replicates, n=50 cilia per sample and repetition. g-h, RPE1 SEPT2-KO cells expressing ARL13B-mRuby2 and Neongreen-EFHC1 after 48h SS and immunostained with Glu-tub antibody. Representative images (g) and quantifications of proximal segment (Glu tub), EFHC1 and whole cilia (ARL13B) length (h) are shown. Three biological replicates, n=100 cilia per sample and repetition. Scale bar: 2µm. i, Time-lapse images of RPE1 SEPT2-KO cells stably expressing ARL13B-mRuby2 and Neongreen-EFHC1. Cells were starved for 32h before the beginning of inspection and imaged every 15min for 9.5h. The point of ectocytosis and the the fragmented cilia are indicated by the white arrow and star, respectively. Scale bar: 5µm. j, Quantification of (i) showing that (f)-ectocytosis mainly occurs at EFHC1-poor region of cilia during time-lapse imaging. Three biological replicates, 50 cilia per sample and repetition.
Figure 3A Whole cell extracts (WCE) were prepared from the indicated strains, immunoprecipitated (IP) with anti-GFP antibody and subjected to western blotting with anti-V5-Pk (Serotec) and anti-GFP (Life Technologies) antibodies. Data are representative of three independent biological repeats.B As for A. C RNA purified from wild type, abo1Δ and spt16-18 cells was analysed by northern blotting using a probe to the 3′ end of spbc19c7.11 (top panel). RNA (5 µg) used for northern blotting was analysed on an ethidium bromide stained 1% TAE agarose gel (bottom panel). Data are representative of two independent biological repeats. D RNA purified from wild type, and abo1Δ cells was analysed by strand specific RT-PCR. RNA from hip1Δ cells was analysed as a control. One primer, complementary to either the forward or reverse transcripts, was included during the reverse transcription step the second primer was then added during PCR amplification. Control reactions omitting the reverse transcription step (-RT) were included to demonstrate the absence of contaminating genomic DNA. Data are representative of two independent biological repeats. E The indicated strains were subjected to ChIP analysis with anti-GFP antibodies and the resulting DNA was analysed by qPCR for the indicated locus. Data are the mean of three independent biological repeats and error bars represent ±SEM. P-values were calculated using a two-tailed unpaired t-test. Significant (P < 0.05) values are indicated. F ChIP analysis of histone H3 levels at act1+ was determined by qPCR. Data are the mean of three independent biological repeats and error bars represent ±SEM. P-value was calculated using a two-tailed unpaired t-test. G The indicated strains were subjected to ChIP analysis as described for E. H Log phase cells were subjected to five-fold serial dilution and spotted onto rich (YE5S) agar or agar supplemented with MMS (0.006%). Plates were incubated for 3-5 days at 30°C or 7 days at 20°C. Images are representative of three independent biological repeats.