EMBO/sd-character-level-ner huggingface dataset:
a, A TRPML1Va-expressing HEK293T cell showed a large inwardly rectifying whole-cell current elicited by voltage steps (from -140 mV to +80 mV in increments of 20 mV) in the standard extracellular (Tyrode’s) bath solution.figf1a, A TRPML1Va-expressing HEK293T cell showed a large inwardly rectifying whole-cell current elicited by voltage steps (from -140 mV to +80 mV in increments of 20 mV) in the standard extracellular (Tyrode's) bath solution. Step duration, 90 ms; holding potential, 0 mV; Vm, membrane potential. b, No significant inward current was detected in NMDG+ (Na+-free, Ca2+-free, pH 4.6) solution. c, Inwardly rectifying step currents were evoked by 30 mM Fe2+ solution (pH 4.6) in the same cell as shown in a and b. d, pH-dependence of IFe/TRPML1Va. Whole-cell currents were elicited by repeated voltage ramps (-100 to +100 mV; 400 ms) with a 4-s interval between ramps. Only a portion of the voltage protocol is shown. Holding potential, 0 mV. e, Large IFe/TRPML2Va was seen in the presence of 30 and 105 mM Fe2+ (pH 4.6). f, Little or no IFe was seen in a TRPML3Va-expressing cell.
Yip1A is responsible for the phosphorylation of IRE1 via high-order assembly of Ire1 molecules.Fig 3HeLa cells were transfected with each siRNA for 24 hr, and then treated with Tm to induce the UPR. Cell lysates were prepared at the indicated time points and analyzed by Western blotting. (A) Representative immunoblots for pIRE1, spliced-XBP1, and β-tubulin. β-tubulin was used for normalization. The intensity of the bands was quantified using the MultiGauge software. (B-D) Relative protein levels of pIRE1 (B), relative mRNA levels of spliced-XBP1 (C), and relative protein levels of spliced-XBP1 (C) in control (solid circles) and Yip1A-knockdown (open circles) cells. The protein or mRNA levels in control cells at the beginning of the Tm treatment were assigned the value 1. Data are means ± SD from three independent experiments. (E) Representative immunoblots for Sar1, Sec23, Sec24D, and GAPDH, and relative protein levels of Sar1, Sec23, and Sec24D in control (solid circles), Yip1A-knockdown (open circles), and IRE1-knockdown (solid gray circles) cells. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the line graphs. The protein levels in control cells at the beginning of the Tm treatment were assigned the value 1. Data are means ± SD from three independent experiments. (F) Representative confocal micrographs of control (upper panels) or Yip1A-knockdown (lower panels) cells during the Tm treatment. Fixed cells at the indicated time points were stained for pIRE1. Cells are outlined with white dashed lines. Scale bars are 10 μm. The numbers of pIRE1 foci per cell were counted, and shown in the line graph. Data are means ± SD (N = 30). (G) Representative immunoblot for pIRE1 after native PAGE, showing two high-order complexes of pIRE1 (pIRE1-I and pIRE1-II). Lane 'S' represents lysate from HeLa cells transfected with control scramble siRNA, and lane 'Y' represents lysate from HeLa cells transfected with Yip1A siRNA. Numbers on the left-hand side correspond to the standard molecular weight. The intensity of the bands was quantified by using the MultiGauge software, and the results are shown in the bar graphs. Protein levels in control cells at the beginning of the Tm treatment were assigned the value 1. Data are means ± SD from three independent experiments. **: p<0.01.
Figure 2Top panel: Y-axis shows the CAGE signal intensity (tag-per-million normalized number of mapped CAGE tags on plus strand). X axis shows LTR and 5' of Tf2 ORF. Three biological replicates are shown for each strain: fft2Δfft3 and WT. Bottom: LTR and tf2 ORF are colored: U3 (green), R (red), U5 (blue), self primer and primer binding sequence (PBS; gray), beginning of ORF (gray striped). TSS of all Tf2 transcripts from (Rhind et al, 2011) are shown as gray dots above the sequence elements. The black square indicates the 5' end of the Tf2 mRNA in WT and the green square indicates the 5' end in fft2Δ fft3Δ detected by 5'RACE.
Figure 2Experimental approach used to image Her6::Venus dynamic expression at single cell resolution. Schematic representation of genetic auto-repression network of Her6 including miR-9 regulation. 3D confocal representative images of a single neural progenitor cell (red arrow) tracked over time, data starting from 34hpf; scale bar 10μm, images from Movie EV3. Single cell time series of Her6::Venus, mKeima-H2B and Her6::Venus signal normalized by mKeima-H2B corresponding to the cell in (c). Covariance models and parameters used to characterize oscillatory (KOUosc ) and non-oscillatory (KOU ) single cell expression. Quantification of single cell dynamics at different stages in normal development including: (f) Proportion of oscillatory cells at different stages in development. (g) period of oscillators estimated using KOUosc , Dashed lines indicate median and dotted lines indicate interquartile ranges of 28hpf (14 cells, 1 embryo), 30hpf (14 cells, 1 embryo) and 34hpf (10 cells, 1 embryo); Mann-Whitney two-tailed test, significance p<0.05 *, p<0.0001****. Quantification of single cell dynamics at different stages in normal development including: (h) noise measured by aperiodic lengthscale (αOU) and (i) local coefficient of variation. Dashed lines indicate median and dotted lines indicate interquartile ranges of 28hpf (14 cells, 1 embryo), 30hpf (14 cells, 1 embryo) and 34hpf (10 cells, 1 embryo); Mann-Whitney two-tailed test, significance p<0.05 *, p<0.0001****.
Both HS and secondary stimuli are required to induce secretion of IL-1β via the NLRP3 inflammasome.Figure 3Representative sections from 9 month old WT and MPSIIIA brains showing secondary storage of GM2 ganglioside, cholesterol, protein aggregates and amyloid beta peptide 1-40 and 1-42 within cortical layers II to V/VI, x20, scale bar: 50 μm. Inserts, 100x, scale bar: 10μm. (n=4 mice per group) ATP levels in whole brain homogenate from 9 month old WT and MPSIIIA mice (n=6 mice per group). Data are expressed as mean ± STDEV and were tested by unpaired t-test. WT vs. MPSIIIA P<0.02 Brain transcription of mRNA species for various inflammasome components (n=6 mice per group). Data are expressed as mean ± STDEV, data for each cytokine were tested by unpaired t-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus WT Caspase 1 activity levels in whole brain homogenate from WT and MPSIIIA mice (n=6 mice per group). Data are expressed as mean ± STDEV and were tested by unpaired t-test. WT vs. MPSIIIA P<0.0001 WT (E-G) and Nlrp3-/- (H-J) mixed glia were primed with MPSIIIA GAG or LPS. ATP (5 mM), cholesterol crystals (1 mg/ml) or amyloid-β fibrils (Aβ1-40 4.3 μM and Aβ1-42 5 μM) were added 4 hours post-priming stimulus, and the secretion of IL-1β into the media measured via ELISA after a further 20 hours (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; *P<0.05, ***P<0.001
ABPs interfaces with actin.Figure 5A Sequence alignment of three actins (Act_Sc, Act_N2 and Act_Ca; deep blue indicates conserved residues, light blue and white indicates non-conserved), indicating contacts between proteins used in the biomimetic assay (with Arp2/3 complex at the mother filament interface (M), with Arp2/3 complex at the daughter filaments interface (D), with tropomyosin (T), with WASP's WH2 (W), with formin (F), with profilin (P), with ADF/cofilin (C), with capping protein (Z), at the protofilament interface (*) and laterally (^). B Schematic representation of actin 3D structure (1YAG (Vorobiev et al, 2003)). Color dots correspond to positions where Act_Sc has different residues compared to Act_N2 (red) and Act_Ca (blue). Purple dots correspond to positions where both Act_N2 and Act_Ca have different residues compared to Act_Sc.
Figure 7Compacted chromosomal interaction domains (red and green) are connected by a domain boundary denoted as a blue line. (Top) High rates of transcription that produce a relatively long (> 2 kb) transcript (wavy orange lines) locally decompact the DNA, thereby creating a spatial gap between flanking loci. This spatial separation diminishes the long-range contacts that can occur within a fixed sphere (black dotted circle), leading to the domain boundaries visible by Hi-C analysis. High rates of transcription can also prevent supercoil diffusion, helping to limit DNA-DNA interactions on a shorter length scale (grey dotted circle). (Bottom) In the absence of transcription, DNA at a domain boundary (blue) will become more compacted, reducing the spatial separation between adjacent domains, and enabling supercoil diffusion, thereby promoting long- and short-range contacts, respectively.
Figure 1EVs were isolated from CCM of uninfected or HIV-1-infected Jurkat cells by differential ultracentrifugation (A-C) (A, C) The successive pellets (2K, 10K, 100K) recovered from the CCM of 20 x 106 cells were analyzed by Western blot for the indicated proteins side-by-side with the lysate of 0.4 x 106 producing cells: C. Images from two different representative experiments are shown. Amount of proteins recovered in 2K, 10K and 100K pellets analyzed in stain-free gel images was similar between pellets: AU (protein amount in a given pellet/total protein amount in 2K+10K+100K) for 2K, 10K and 100K pellets were 1.13±0.28, 0,96±0.10 and 0.91±0.33, respectively in control cells (A), and 1.22±0.29, 0,84±0.35 and 0.94±0.30, respectively in infected cells (C) (mean±SD). EVs were isolated from CCM of uninfected Jurkat cells by differential ultracentrifugation SD). (B) Whole-mount EM analysis showing representative images and size distribution of particles in 2K, 10K and 100K pellets (Scale bar, 200 nm). The diameter (nm) of particles was determined with ImageJ for five different experiments. 387, 363 and 385 particles were counted for 2K, 10K and 100K pellets, respectivelly. (A-D) EVs were isolated from CCM of uninfected or HIV-1-infected Jurkat cells by differential ultracentrifugation and sEVs contained in the 100K pellet were further separated by velocity iodixanol gradient (D). (D) 12 fractions were recovered from iodixanol gradients of the 100K pellets obtained from CCM of non-infected and infected Jurkat cells and analysed by Western blot for the indicated proteins.
Fission is associated with bioenergetic failure and free radicals.Figure 6(A) Mitochondrial fission is linked to a drop in ATP. Purified corticalneurons were exposed to increasing SNOC concentrations or mitochondrial inhibitors (2 μM rotenone plus 2 μg/ml oligomycin; Rot/Olig). ATP concentrations are shown as the mean±s.e.m. normalized to the plating density of neurons (n=6) (B) [ATP]/[ADP] ratios in purified corticalneurons exposed to increasing SNOC concentrations (n=4). (C) The bar graph depicts ethidine fluorescence density in arbitrary units of aged and fresh SNOC treated neurons at 2.5 h. (D) Cortical neurons expressing Mito‐GFP (green) were exposed to aged or fresh 200 μM SNOC solution. After 2.5 h cultures were loaded with hydroethidine. Images are representative of more that 20 neurons analyzed (in D) for each condition from at least three independent experiments. Scale bar, 10 μm. (E) Reduced glutathione (GSH) partially blocks mitochondrial fission. Neurons were transfected with Mito pretreated for 2 h with 2 mM GSH monoethyl ester, washed once and then exposed to 35 μM SNOC. Mitochondrial fission was scored after 1 h. Data are means±s.e.m. of quintuplicate samples from one representative experiment of a total of three (†††significance at P0.001 or n.s., †not significant compared to control; *significance at P0.05).
Figure 10A Top, representative photographs of 20-week-old male ob/ob and FADD-D/ob/ob mice fed a SD. Bottom, representative photographs of their livers and epididymalWAT. B Comparison of weights of WAT depots from WT, FADD-D, ob/ob and FADD-D/ob/ob mice. n = 5 for each genotype. Data are expressed as mean ± SEM. *P = 0.0007, **P = 0.0022, ***P = 0.0011, #P = 0.0009, ##P = 0.0003, ###P = 0.0027 (one-way ANOVA). C Basal and stimulated lipolysis measured by fatty acid release from explants of epididymalWAT in 10-week-old male ob/ob and FADD-D/ob/ob mice fed a HFD. n = 4 for each genotype. Data are expressed as mean ± SEM. *P = 0.0003, **P = 0.001 (one-way ANOVA). D cAMP level in WAT of 10-week-old male WT, FADD-D, ob/ob and FADD-D/ob/ob mice fed a HFD. n = 4 for each genotype. Data are expressed as mean ± SEM. *P = 0.0017, **P = 0.001 (one-way ANOVA). E Whole-body oxygen consumption rate (VO2) during 24 hr in WT, FADD-D, ob/ob, and FADD-D/ob/ob mice fed a SD. n = 4 for each genotype. Data are expressed as mean ± SEM. *P = 0.0053, **P = 0.0025, ***P = 0.0062, ****P = 0.0011 (one-way ANOVA). F Quantitative real time PCR for Dio2, PPARd and UCP1, using RNA from epididymal fat from 20-week-old male ob/ob and FADD-D/ob/ob mice fed a SD. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0009, **P = 0.0015, ***P = 0.0003 (Student's t-test). G Quantitative real time PCR for UCP3, PGC-1α, PPARd and CPT1, using RNA from skeletal muscle from 20-week-old male ob/ob and FADD-D/ob/ob mice fed a SD. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0105, **P = 0.0033, ***P = 0.0058 (Student's t-test). NS, not statistically significant. H Quantitative real time PCR for UCP1, PPARa, CPT1 and PGC-1α, using RNA from brown adipose fat from 20-week-old male ob/ob and FADD-D/ob/ob mice fed a SD. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0012 **P = 0.0129, ***P = 0.0103, ****P = 0.0207 (Student's t-test). I Oxidation of [1-14C] oleic acid to 14CO2by adipocytes isolated from WT, FADD-D, ob/ob, and FADD-D/ob/ob mice. Data shown are from one experiment (n = 3 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0031, **P = 0.0075 (one-way ANOVA). NS, not statistically significant. J Immunoblot of VLCAD, LCAD, MCAD, PTL and Tubulin (control), using protein extracts from epididymal fat from 15-week-old male WT, FADD-D, ob/ob and FADD-D/ob/ob mice fed a SD. Data shown are representative of three independent experiments having similar results.
Figure 5HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3, Flag-ABRO1 and HA-ubiquitin (HA-Ub), HA-Ub K48 only or K63 only. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose. HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding HA-Ub, Flag-ABRO1, and NLRP3 truncations as indicated. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of ubiquitination of NLRP3 truncations (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose. LPS-primed Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP (left) or nigericin (right). Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody. Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were left unstimulated or stimulated with LPS or LPS plus ATP. Immunoblot analysis of NLRP3 and NLRP3 (A350V) ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads. HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3 (A350V) and HA-ubiquitin (HA-Ub). Immunoblot analysis of NLRP3 (A350V) ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads. Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were stimulated with or without 100ng/ml LPS for 1 h. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody. HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3, and HA-ubiquitin (HA-Ub) were left untreated or treated with 5 μM Anisomycin for 2 h or 100 nM SP600125 for 2 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads. LPS-primed WT and Nlrp3S194A/S194A BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.
Figure 6The expression of LIMT is shown relative to (A) PAM50 subtypes (N=381 patients), (B) tumor grade (N=309), (C) HER2 status (N=309) and (D) ER status (N=381 patients). Expression of individual lncRNAs was determined in tumors from breast cancer patients of the Oslo2 study using Agilent arrays. Molecular subtypes of the corresponding tumors were determined by using the PAM50 classifier. The Student's t-test was applied to evaluate differences in lncRNA expression between two groups. To evaluate differences in expression among three or more groups, we applied one-way analysis of variance (ANOVA).
a) Atg7+/− mice were generated as described in the Methods.f1(a) Atg7+/− mice were generated as described in the Methods. PCR was done employing genomic DNA and primers flanking the floxed Atg7 regions. Arrows indicate wild-type Atg7 band in Atg7+/+ mice, increased size of PCR band due to inserted flox sequence in Atg7F/+ mice and decreased size of PCR band due to Cre-mediated deletion in Atg7+/− mice, respectively. (b) RT-PCR (left) and real-time RT-PCR (right) were performed using total RNA prepared from tissues of Atg7+/− or control Atg7+/+mice, and primers specific for Atg7 or β-actin sequence. Fold changes of the RT-PCR band intensities are shown (left). *P0.05, **P0.01, ***P0.001; Student's t-test, n=3. (c) Tissue lysate from fasted 12-week-old Atg7+/− and control Atg7+/+mice was subjected to immunoblot analysis using anti-LC3 or -p62 antibody. Fold changes of the immunoblot band intensities are shown (middle and right). **P0.01, ***P0.001; Student's t-test, n=3. (d) Cell lysate of Atg7+/+, Atg7+/− and Atg7−/− MEFs was subjected to immunoblot analysis using antibodies specific for LC3 and p62 after rapamycin (Rap) or control solvent (dimethyl sulfoxide, DMSO) treatment for 3 h. Fold changes of the immunoblot band intensities are shown (middle and right). *P0.05, **P0.01, ***P0.001; Student's t-test, n=3. (e) Sections of the liver and muscle tissues from fasted 12-week-old GFP-LC3+-Atg7+/+ and GFP-LC3+-Atg7+/− mice were subjected to fluorescent microscopy to examine GFP-LC3 puncta (left). The number of GFP-LC3 puncta was counted (right). Scale bars, 10 μm. *P0.05; Student's t-test, n=5. (f) Tissue lysates were prepared from the liver of fasted Atg7+/+ and Atg7+/− mice 4 h after leupeptin administration, and immunoblotting was done. Fold changes of the immunoblot band intensities are shown (right). *P0.05; Student's t-test, n=3.
Effect of liraglutide and RM-493 co-treatment on energy metabolic parameters in DIO mice.Figure 2A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (A, B) energy expenditure. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001. A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (C) respiratory exchange ratio (RER). Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001. A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (D) locomotor activity. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001. A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (E) cumulative food intake, (F) meal number and (G) meal size. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.
Stable PINK1 knockdown in SH-SY5Y cells.Figure 1A) SH-SY5Y neuroblastoma cells were either cultivated in RPMI+10% FCS or starved in HBSS with or without addition of LY294002 or rapamycin. After 16 h PINK1 mRNA expression was determined by RT-qPCR and the PINK1 mRNA content of cells kept in RPMI+10% FCS was set as 1. Induction of autophagy by starvation or rapamycin resulted in an induction of PINK1 expression, comparable to the positive control LY294002; n = 4; * expression changes compared to the PINK1 expression in RPMI+10% FCS: LY294002: p<0.01; rapamycin: p<1×10−4; HBSS: p<0.005; HBSS+LY294002: p<5×10−6; HBSS+ rapamycin: p<0.0005; # expression changes compared to the PINK1 expression in RPMI+10% FCS+LY294002: p<0.05; + expression changes compared to the PINK1 expression in RPMI+10% FCS+rapamycin: p<0.0005. B) SH-SY5Y cells were either stably transduced with a control (nt) shRNA or a shRNA directed against PINK1 and cultivated in RPMI medium containing 5% or 10% FCS. Their PINK1 mRNA content was determined by RT-qPCR and PINK1 mRNA content of nt cells kept in medium with 10% FCS was set as 1. Serum reduction increased PINK1 mRNA in nt cells in accordance with the data shown in Fig. 1A, while stable PINK1 knockdown (kd) reduced PINK1 content under both conditions; n = 3; * expression changes compared to the PINK1 expression in RPMI+10% FCS: PINK1 kd 10% FCS p<0.0005; PINK1 induction by 5% FCS: p<0.05; # expression changes compared to the PINK1 expression in RPMI+5% FCS: PINK1 kd 5% FCS: p<0.005. C) SH-SY5Y cells without (nt) or with stable PINK1 knockdown (kd) were kept in medium with 5% FCS and either untreated or treated for 2 h with CCCP to stabilize PINK1. Afterwards the PINK1 63 kDa protein (arrowhead) and actin protein levels were determined by western blotting (see representative gel on the right). The quantification revealed a reduction of PINK1 protein under both conditions in PINK1 kd cells.
Benomyl sensitivity of wild‐type cells overexpressing AUT2 and AUT7, and of aut2Δ and aut7Δ cells is not altered.Figure 6Benomyl sensitivity of wild‐type cells overexpressing AUT2 and AUT7, and of aut2Δ and aut7Δ cells is not altered. Cells were grown to stationary phase and dilution series with decreasing cell densites were dropped on CM plates without uracil containing 15 and 30 μg/ml benomyl. Plates were incubated at 30°C.