EMBO/sd-character-level-ner huggingface dataset:
Figure 6Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 12hr glucose starvation (Glu-) Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 36hr hypoxia (B). (C) Western blot detection of CHK2, HSP60, TOM20 and TIM23 in H1299 cells transfected with the indicated shRNA in normal medium or after12 hr glucose starvation (Glu-) or 36 hr hypoxia. (D-E) Transmission electron microscopy of indicated H1299 cells cultured in glucose starvation (Glu-) medium or hypoxia. The yellow asterisks mark mitochondria. The red arrows indicate autophagic structures. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; ***P < 0.001 compared to shNC treated with H2O2 or hypoxia (Mann-Whitney test); Scale bar, 1 μm. (F) Representative confocal images of the shift in the fluorescence emission of Keima from 458 to 561 nm. Scale bar, 10 μm. The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or after 18 hr glucose starvation (G) All results are from three independent experiments. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 (Mann-Whitney test). The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or 48 hr hypoxia (H). All results are from three independent experiments. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 (Mann-Whitney test).
Figure 2(A) Domain diagram of crystallized Hrr25 constructs from S. cerevisiae and C. glabrata, missing the P/Q-rich C-terminal domain. (B) Structures of S. cerevisiae Hrr251-394 K38R bound to CK1-7 (Chijiwa et al, 1989; Xu et al, 1996) and C. glabrata Hrr251-403 K38R bound to ADP, with domains colored as in (A). Bound PO4-/SO4- ions are shown as spheres. See Figure EV2A for additional C. glabrataHrr25 structures, and Figure EV3A for SO4- ion electron density. (C) Overlay of the kinase domain of C. glabrataHrr25 (Apo form, crystallized with 1.2 M PO4-) with ratCK1δ crystallized in the presence of tungstate ions (WO4; PDB ID 1CKJ, (Longenecker et al, 1996)) See Figure EV2B for detailed views of ion binding. (D) Stereo view of CK1-7 binding to S. cerevisiae Hrr25. Bound drug is positioned identically to a previous structure of S. pombe Cki1 bound to CK1-7 (Xu et al, 1996). (E) Stereo view of ADP binding to C. glabrata Hrr25 (formate structure; SO4- structure is equivalent, but the GxGxxG motif is disordered in that structure). All active-site residues shown in panels (D) and (E) are conserved between S. cerevisiae and C. glabrata Hrr25.
Figure 3(A) Effect of lactate on the ERK/TSC2 and AKT/TSC2 signaling pathway in serum-starved HCT116 cells. (B) Effect of lactate on phosphorylated S6K (T389) inhibited by TSC2 overexpression in HCT116 cells. (C) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA-Rheb-overexpressing HCT116 cells after serum deprivation overnight. (D) Molecular interaction between TSC2 and recombinant GDP-loaded Rheb in the presence or absence of lactate in vitro. (E) Effect of lactate on Rheb activation in serum-starved HCT116 cells. (F) Interaction between endogenous Rheb and mTOR in the presence or absence of lactate in HCT116 cells after serum deprivation overnight. (G) Immunofluorescence analysis of staining for TSC2 and LAMP2 in the presence or absence of lactate in HCT116 cells after serum deprivation overnight.
Figure 5HME control (vec) and OAS single knockout cells (KO1-3), pre-treated with IFNβ for 2 days to induce OAS expression, fixed, and stained with anti-OAS1, 2, or 3. Staining with secondary antibodies is shown as a negative control. Scale bar, 10μm. HME control cells were treated with IFNβ for 2 days to induce OAS expression, fixed and stained with anti-OAS1, 2, or 3. Scale bar, 10μm. HME cells expressing Flag-tagged OAS1, 2, or 3 were fixed and stained with anti-Flag. Scale bar, 10μm. Untreated HME (left) or PC3 (right) cells were stained with anti-OAS1-3 and the nuclei were visualized by staining with DAPI. The intensities of OAS1-3 fluorescence signals in the nuclei are plotted. Nuclear and cytoplasmic masks were made based on DAPI and phalloidin staining, respectively. Experiments were repeated at least two times; at least 40 cells per condition were counted. Data are presented as distribution plot with mean value +/- SD; statistical significance was determined by unpaired Kolmogorov-Smirnov t-test. Scale bar, 10μm. HME control (vec) and KO1 cells were pre-treated with IFNβ for 2 days, followed by a 10-min treatment with H2O2. Cytoplasmic, membrane-bound and soluble nuclear fractions of cell lysates were analyzed by the Western method; staining with anti-AIF and anti-HDAC2 staining was used to characterize membrane and nuclear fractions, respectively. Anti-PAR detects all PAR-modified proteins, which appear as smears. HME control (v), KO1 and OAS1hi (O1hi) cells were pre-treated with IFNβ for 48 h. Cytoplasmic, membrane-bound and soluble nuclear fractions of cell lysates were analyzed by the Western method. Anti-AIF and anti-HDAC2 were used to characterize membrane and nuclear fractions, respectively; * indicates lanes where specific signals are detected.
Figure 3A Time lapse confocal images of PHcrac (top) and LimE (bottom) in the same giant cell (scale bar, 20 μm). White arrows point to wave breaking events. B Three phase plane representations of the activator (F) - inhibitor (R) system with the activator nullcline (red) and the inhibitor nullcline (brown) for different inhibitor levels. The horizontal dashed line corresponds to the initial inhibition level (R0) at equilibrium (blue circle). The net area (rightmost plot) is calculated by adding the positive area above the horizontal line (dark red shade) and the negative area below (light red shade), for increasing levels of inhibition (R0 = Rlow, Rhigh and Rstop). The points Rlow and Rhigh correspond to high (chigh) and low (clow) wave velocities, respectively, as indicated on the right. C (Left) Kymograph plotting the inhibitor activity with time and space as axes. The point where the wave is triggered is indicated in yellow. (Right) Two line-scans (i and ii) from the kymograph and the corresponding activity levels. R0 indicates the inhibition level at the time of the first trigger, while Rstop indicates the critical stopping threshold. The activator profile is shown via the dashed red plot while the inhibitor is the solid brown line. The grey vertical dashed line marks the point when the activator element is triggered. The black horizontal dashed line denotes the inhibition level at the time of the specific trigger shown. D Kymographs corresponding to high (left) and low (middle) thresholds with the white arrow denoting the range of the wave, which is calibrated with threshold R0 (right). The high threshold wave is superimposed in yellow on the low threshold wave to emphasize the increase in wave velocity as the low threshold wave spreads further in space compared to the high threshold wave. E Two-dimensional simulations where two waves, initiated at different thresholds, are triggered at the bottom left corner (leftmost panel). Overlap between the two waves appears as yellow. The panels show the evolution of the two waves with time.
Figure 5A) Transient co-expression of MKKK7 in Arabidopsis mesophyll protoplasts shows suppression of flg22-induced WRKY29 gene expression. Protoplasts were transfected with pWRKY29:fLUC, 35S:rLUC and indicated overexpression constructs of MKKK7 (OE-MKKK7, OE-MKKK7AA or OE-MKKK7DD) as indicated on the horizontal axis. Protoplasts were treated with 10 μM flg22 or mock treated for 4 hrs. All measurements were normalized to the rLUC activity and expression is relative to the mock treated control sample, shown as fold induction on the vertical axis. Results shown are means ± STDEV (n=2). At least two biological replicates were done with similar results B) Protein structure of MKKK7 and mutated versions of MKKK7 with the protein kinase domain shown in yellow and an ARM/HEAT repeat domain shown in blue. The position of the phosphorylated Serine residues is indicated with triangles and bold S below the protein structure. The red triangles indicate phosphorylated Serines that were targeted for mutagenesis or the corresponding phospho-mimic Aspartic acid. Blue triangles indicate the substitution with the non-phosphorylatable amino acid Alanine. Amino acid substitute versions of MKKK7 are shown below the wild-type. S, Serine; A, Alanine; D, Aspartic acid.
Rab7 interactions influence SKIP-associated HOPS complex assembly.Figure 9Effects of SKIP KMI motif on HOPS complex recruitment. A. Analysis of SKIP/HOPS interactions by co-IP of RFP/mCherry-HOPS subunits with Myc-SKIP versus the KMI motif mutant AAA from HEK293T cells using RFP-trap beads. Representative immunoblots against RFP and Myc are shown, IP: immunoprecipitation, TL: total lysate. B. Quantification of SKIP/HOPS interactions, n=3 independent experiments. Top graph: fold change in binding of HOPS subunits to AAA mutant relative to wild type (WT) SKIP (1.0), significance: one-way ANOVA test (relative to VPS41). Bottom graph: Co-IP per HOPS subunit relative to VPS39, significance: one-way ANOVA test (relative to VPS39), * p<0.05, *** p<0.001, ns: not significant. C. Colocalization (Mander's overlap) between SKIP and RFP-VPS39 (images or mCherry-VPS41 (images , n≥10 images (2≥ cells per image) analysed per condition from 2 independent experiments. D. Representative confocal images of fixed HeLa cells ectopically expressing Myc-SKIP, KMI motif mutant (AAA) or RUN domain truncation mutant (∆RUN) (red) together with RFP-VPS39 (green), immunolabeled for endogenous VPS11 (eVPS11, blue). Effect of HOPS over-expression on recruitment of TBC1D15 to the SKIP compartment. E. Representative confocal images of fixed HeLa cells harbouring endogenous GFP-TBC1D15 (Ge1D15, green) and ectopically expressing HA-SKIP (red) together with mCh-VPS18 or RFP-VPS39 (white), immunolabeled for endogenous VPS18 or VPS11 (blue), as indicated F-I. Colocalization of SKIP and HOPS complex with TBC1D15 as a function of HOPS subunit over-expression. F/H. Mander's overlap between F. SKIP or H. eVPS18 and endogenous TBC1D15, n≥5 images (2≥ cells per image) analysed per condition from 2 independent experiments, significance: one-way ANOVA test (relative to EV), ** p<0.01, *** p<0.001, ns: not significant. G/I. Mander's overlap between G. SKIP or I. eVPS11 and endogenous TBC1D15 as a function VPS18 overabundance, with or without FIS1 depletion (siFIS1), n≥3 images (2≥ cells per image) analysed per condition from 2 or more independent experiments.
Decreased grip strength, increased denervation, and increased expression of denervation and atrophy markers throughout disease progression in SOD1G86R mice.Figure 2Grip test was performed to measure mousemuscle strength at different time points during disease progression. Graph represents the mean of percentage from initial force measured at 11 weeks ± SEM. At 13 weeks, *P = 0.03; at 14 weeks, P = 0.06; at 15 weeks, *P = 0.04 (n = 8 for WT and SOD1G86R, respectively, at 11-15 weeks, two-way ANOVA followed by Fisher's LSD post hoc test). Electromyography (EMG) recordings were performed weekly during disease development, starting from 9 weeks of age. Spontaneous electrical activity was considered as positive EMG only for peak-to-peak amplitudes > 50 μV. Left: The percentage of mice presenting with positive EMG in each age group are represented. Right: Representative examples of negative (EMG−) and positive (EMG+) electromyography profiles (n = 10 for WT and SOD1G86R, respectively, at 9-13 weeks and n = 8 and 7 for WT and SOD1G86R, respectively, at 15 weeks). Relative mRNA levels of denervation markers AChR (subunits α and γ) and MuSK were evaluated by qPCR at the indicated ages (65 and 105 days) in tibialis anterior (upper panels) and soleus (lower panels) of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT (n > 5). ***P-values versus WT: < 0.0001 for AChRα, AChRγ, and MuSK in tibialis anterior; and **P-values versus WT: 0.001 for AChRα, and ***P-values versus WT: 0.0007 for AChRγ, 0.0002 for MuSK in soleus (n=7 and 8 for WT and SOD1G86R, respectively, at 65 days; n = 5 and 6 for WT and SOD1G86R, respectively, at 105 days; two-way ANOVA followed by Fisher's LSD post hoc test). Relative mRNA levels of muscle atrophy markers MuRF1 and Atg-1 were measured by qPCR at the indicated ages (65 and 105 days) in tibialis anterior (upper panels) and soleus (lower panels). Graphs represent mean fold change ± SEM from age-matched WT. ***P-values versus WT: < 0.0001 for MuRF1 and Atg-1 in tibialis anterior; and **P-values versus WT: 0.007 for MuRF1 and P = 0.006 for Atg-1 in soleus (n = 7 and 8 for WT and SOD1G86R, respectively, at 65 days, n = 5 and 6 for WT and SOD1G86R, respectively, at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).
Figure 2Circles represent metabolites and connecting lines indicate metabolic reactions. Metabolite names are indicated in black, while reaction names and associated gene names are in dark blue. Reaction directionality is represented by arrows. The dotted line shows the cell membrane, with transport reactions linking the intracellular milieu with the extracellular environment. The reactions are color-coded according to the six main modules. The outer colored boxes describe the number of genes, gene-associated reactions, and orphan reactions. An interactive Escher (King et al, 2015) version of this map is available (Computer Code EV1).
Figure 2A Effect of KDM6A knockdown on the expression of nephrin and WT-1 in immortalized mouse podocytes cultured under high glucose conditions. Western blot analyses were performed using protein lysates of podocytes that were transfected with scrambled siRNAs or KDM6A-specific siRNAs, and cultured in high glucose (HG) or high mannitol (Mann) for 48 hours. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with HG incubation (Parametric ANOVA and a Bonferroni post hoc test; n = 3). B Effect of KDM6A overexpression on expression levels of nephrin and WT-1. Immortalized podocytes were transfected with the KDM6A expression plasmid or the empty vector control for 48 hours, and the expression levels of KDM6A, nephrin and WT-1 in these transfected podoytes were analyzed by immunoblotting. *P < 0.05 versus vector controls (Parametric ANOVA and a Bonferroni post hoc test; n = 3). C Changes in H3K27me3 levels in podocytes after treatment with high glucose, the combination of high glucose and KDM6A knockdown, or KDM6A overexpression for 48 hours. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with HG incubation (Parametric ANOVA and a Bonferroni post hoc test; n = 3). D Changes in DNA methylation at nephrin gene promoter by high glucose, by the combination of high glucose plus KDM6A knockdown, or by KDM6A overexpression in immortalized podocytes. U, unmethylated-specific primers; M, methylated-specific primers. Experiments were repeated three times and a representative gel from one experiment is shown. E Effect of GSK-J4 (40 μM), a specific KDM6A inhibitor, on high glucose-mediated changes in levels of nephrin, WT-1 and H3K27me3. *P < 0.05 versus normal controls, #P < 0.05 versus untreated HG-incubated cells (Parametric ANOVA and a Bonferroni post hoc test; n = 3). F Changes in DNA methylation at nephrin gene promoter by GSK-J4 (40 μM) in immortalized podocytes. U, unmethylated-specific primers; M, methylated-specific primers.
Nrl.Figure 2a, Schematic representation of photoreceptor transplantation, shown in (b); b, (left) P8 Nrl.Cre+/- photoreceptors transplanted into subretinal space of TdTomatofloxed reporter mice (N = 5 eyes), compared to (middle) subretinal injection of AAV-Nrl.Cre subretinal injection (10^12 vp/ml), (N = 10 eyes) and (right) contralateral uninjected control (N = 10 eyes). Arrows indicate recombined host photoreceptors (white arrows) or RPE (green arrows). Note clearly recombined RPE cells upon AAV transduction. c, schematic representation of subretinal EV injection, shown in (d). Red indicates host cells undergoing Cre-mediated recombination and expressing TdTomato (red); d, P8 Nrl.Cre+/- PR -derived (left) 100K, (middle) 10K and (right) 2K EV pellets injected into the subretinal space of TdTomatofloxed reporter mice (N = 4 eyes per EV preparation). Recombination was restricted to Müller Glia (asterisks). e, schematic representation of intravitreal EV injection, shown in (f); f, (left) intravitreal injection of P8 Nrl.Cre+/- photoreceptor-derived 100K EV pellets (N = 2 eyes). Recombination was restricted to Müller Glia (asterisks) with little or no recombination in either photoreceptors or the RPE; (right) subretinal injection of recombinant Cre protein (control) into TdTomatofloxed reporter mice. Blue = nuclei, red = TdTomato+ve recombined cells. N = 4 retinae per group. Representative MIP confocal images; g, Quantification of no. of TdTomato+ve host photoreceptor cells seen following transplantation of live (224 ± 181) versus UV-treated (dead) (3 ± 4) Nrl.Cre+/- P8 photoreceptors (N = 4 eyes per condition). Graph shows mean ± S.D.
RBM14 can suppress centriole amplification in cells arrested in S phase.Figure 2A U2OS cells or U2OS cells expressing FLAG-RBM14 full length (FL, aa1-669), N-terminal fragment (RBM14[N], aa1-150) or C-terminal fragment (RBM14[C], aa151-669) and treated with control siRNA or siRNA against 3′ UTR targeting endogenous RBM14 were stained with antibodies against FLAG as well as centrin-2. Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition. Values are mean percentages ± SEM from three independent samples (n = 30 for each condition). **P < 0.01, n.s., not significant (one-tailed t-test). B Schematic of full-length, truncated mutants and NES-fused and PACT-fused RBM14 proteins used in this figure. C, D Cytoplasmic RBM14 could suppress centriole amplification in HU-treated cells. (C) U2OS cells or U2OS cells expressing FLAG-RBM14 FL or [C] and treated with or without HU were stained with antibodies against centrin-2 (green) and FLAG (magenta). (D) U2OS cells or U2OS cells expressing FLAG-RBM14 FL, FLAG-RBM14-NES or GFP-RBM14-PACT and treated with or without HU were stained with antibodies against FLAG (magenta, left) or GFP (magenta, right) as well as centrin-2 (green). Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 10 μm. Histograms represent frequency of cells in interphase with excess centrin foci in each condition. The percentages of U2OS cells with centrosomal localization of the RBM14 full-length protein or mutants are shown below the immunofluorescence images. We counted only cells that had adequate intensity of FLAG or GFP signals and did not find any significant difference in the total expression levels of the exogenous RBM14 proteins. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test). Please note that cytoplasmic expression levels of GFP-RBM14-PACT are less than those of FLAG-RBM14 FL (˜0.5-fold) or FLAG-RBM14-NES (˜0.7-fold).
Figure 6A) Principal component analysis of the DMDMex RNA-seq counts. B) Volcano plot for the F test on differential expression between healthy individuals and DMD patients. C) Scatter plot showing the group effect at baseline (x-axis) and the effect of the interaction between age and group (y-axis). Black dots represent non differentially expressed genes, while blue dots represent differentially expressed genes. D) Venn diagram showing the overlap between the genes identified as differentially expressed in DMD patients E) Volcano plot for the F test on differential expression between treated and untreated DMD patients. F) Barplot comparing the logFC of the genes associated with steroid treatment estimated in this study to the estimates of Liu et al. (2012). G) Scatter plot showing genes for which a significant effect of treatment with steroids. The group effects at baseline are plotted on the x-axis, while the effects of steroids are plotted on the y-axis. H) Heatmap obtained by Ingenuity Pathway Analysis comparing the pathways affected in DMD patients compared to the pathways affected in mice at the different time points. I) Correlation circle representing the correlation between physical tests and their corresponding first two principal components. J) Scatter plot showing the relationship between the first principal component of body measurements and the expression levels of LAPTM4B.
Electroporated-antibody dependent neutralization assay (EDNA) for coronavirus.Figure 1(A) A titration of anti-MHV polyclonal serum was added either directly to media (extracellular) or electroporated into L929 cells (intracellular), then cells were infected with MHV-A59. The kinetics of cell growth was monitored by measuring total cell area. Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates. (B) Phase-contrast images of cells at 48h post-infection. Scale bar = 200µm. (C) At 48h post-infection, the viability of cells infected with MHV-A59 in the presence of intracellular or extracellular antiserum was determined by ATP luminescence assay. Increasing doses of antiserum results in increased cell survival. Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates. (D) Kinetics of cell growth following MHV-A59 infection in the presence of a titration of electroporated anti-N antibody. Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates. (E) Phase contrast images of cells at 48h post-infection. Scale bar = 200 µm. (F) Quantification of cell viability by ATP luminescence assay at 48h post-infection in the presence of electroporated anti-N or anti-GFP antibodies. Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates. (G&H) WT or TRIM21 KO L929 cells were infected with MHV-A59 in the presence of electroporated polyclonal antiserum (G) or anti-N antibody (H) and quantified by cell area 48h later. Co-electroporation of Protein A/G with anti-N antibody into WT cells mimics the TRIM21 KO phenotype. Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates. (I) L929 Cells were infected with MHV-A59 for 4h, then cyclohexamide (CHX) was added to block further viral protein synthesis. After 5h, cells were electroporated with anti-N or anti-GFP antibodies and left for a further 3h before being western blotted for cellular N protein levels (*denotes a non-specific band). Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates.
Figure 4Venn diagram depicting the common interacting proteins between different tsRNAs in RA treated cells. Scatter plot showing Log2 fold-change enrichment of protein interactome between LIF- and RA-treated mESCs. All peptides identified by LC-MS/MS had < 1% FDR (Fig EV4E). LC-MS/MS was conducted twice (R2 = 0.85 between LIF duplicates and R2 = 0.82 between RA-treated mESCs duplicates). Scatter plot showing Log2 fold-change enrichment of mRNAs associated with tsGlnCTG over Scramble (Scr1) in LIF- versus RA-treated mESCs. Bar plots and representative gene lists depicting association of tsGlnCTG with 'pluripotency-associated' (D) and 'differentiation-responsive' (D') genes in LIF- or RA-treated mESCs.