EMBO/sd-character-level-ner huggingface dataset:
Figure 3The regulatory mechanism of the rDNA transcription. Firstly, UBF1 binds to the upstream control element (UCE) of rDNA promoter to recruit SL1, then SL1 recruits Pol I for rDNA transcription. Co-IP analysis of nucleolar proteins with NF90 in HEK293 cells transfected with EV, and the following plasmids: GFP-S5, GFP-L9, GFP-B23, GFP-UBF, GFP-C23. * represents the target protein. Co-IP analysis of endogenous UBF with NF45 and NF90 in HEK293 cells. HEK293 cells were infected by lentivirus containing NF45-specfic shRNA or NF90-specfic shRNA, and then stimulated in serum for 0, 3 and 6 h. Cell lysates were prepared to analyze the expression levels of the indicated proteins by western blot. ChIP analysis was used to determinate the binding ability of NF45 and NF90 to rDNA promoters in HeLa cells stimulated with serum for 0, 3 and 6 h. (n = 3) ChIP analysis of NF45 and NF90 binding to the H42.9 loci in HeLa cells with the treatment of CX5461 or DMSO for 2 h. (n = 3) Confocal images of HeLa cells treated with CX5461 for 2 h showing NF45 and NF90 colocalized with fibrillarin. Scale bar, 10 μm.
Figure 3A) Venn diagram showing the overlap of sub-nucleosomal MSL2 DNA peaks and mono-nucleosomal MSL2 chromatin regions. B) Average composite plots of MSL2 [Chromatin] profiles (stage 5-8, orange and stage 13-15, purple) centered at MSL2 DNA peaks which do not overlap (DNA) or overlap MSL2 chromatin regions (DNA + Chromatin), and MSL2 chromatin regions which do not overlap MSL2 DNA peaks (Chromatin). Graphs represent average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites. C) Average composite plots of MSL2 [Chromatin] profiles centered at MSL2 chromatin regions called in both early and late (left) or only in late stages (right). Graphs represent average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across regions. Total number of early = 212 and late = 87. D) Average composite plots of MOF [Chromatin] profiles centered at MSL2 chromatin regions called in both early and late (left) or only in late stages (right).
A N-terminal Nup50 fragment is required for NPC assembly (A) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with recombinant wild-type Nup50 or different mutants.Figure 6(A) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with recombinant wild-type Nup50 or different mutants. Nuclei were fixed in 4% PFA and 0.5 % glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. (B) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (A). Data points from the individual experiments are indicated. (C) Confocal microscopy images of nuclei assembled with N-terminal Nup50 truncations and an minimal N-terminal fragment. Samples were analyzed as in (A). (D) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (C). Data points from the individual experiments are indicated.
Figure 3A, Example images of hippocampal neurons co-transfected with Mito-mKate2 and VGlut1pHluorin before and during field stimulation. The white arrow indicates a terminal occupied with a mitochondrion, while the empty arrow indicates a terminal without a mitochondrion. B, Average traces of presynaptic terminals of hippocampal neurons transfected with VGlut1pHluorin and Mito-mKate2 and stimulated using field stimulation at 10 Hz for 20s (40-60s). Terminals with a mitochondrion are represented by the red trace and terminals without are represented by the black trace. C, Summary graph corresponds to the traces shown in panel B). Mean stimulation fluorescence (time points 60-70s) comparing terminals of the same axon which contain mitochondria (right, ΔF/F0 = 2.3 0.9) compared to terminals without mitochondria (left, ΔF/F0 = 3.2 0.7) (n=9 neurons, 66 terminals, *p<0.05, paired t-test). Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.
The Rb−/− muscle defect is characterized by perinuclear mitochondrial/autophagosome aggregates, reduced mitochondrial DNA content, and reduced ATP production.Figure 2.(A, left) MitoTracker staining. (right) MitoTracker staining after bezafibrate treatment. (B) Quantification of myotubes with perinuclear MitoTracker-positive aggregates in untreated (vehicle) or bezafibrate-treated myotubes at DM-2. Data represent mean ± SD (n = 3). *, P < 0.001 by Student's t test. (C) MitoTracker (red) and LC3immunostaining (green) of myotubes at DM-2. Chloroquine and 3-MA (where indicated) were added 12 h before staining. Note the extensive overlap of MitoTracker and LC3 in Rb−/−myotubes (yellow), which is inhibited by 3-MA. (D) Mean percentage of MitoTracker (red) to LC3 (green) overlap in 25 myotubes from two independent cultures in control versus Rb−/−myotubes (total) or Rb−/−myotubes with perinuclear mitochondrial aggregation (high). *, P = 7 × 10−5 by Student's t test. (E) Ultrastructural analysis of myotubes treated or not with 3-MA at DM-2. Arrowheads point to mitochondria. Asterisks indicate electron-dense autolysosomes. N, nucleus. (F) Ratio of mitochondrialDNA/nuclearDNA in the indicated cultures (n = 3). (G) Ratio of mitochondrialDNA/nuclearDNA in E16.5 skeletal muscle (n = 3; *, P = 0.009). (H) Quantification of ATP in E16.5 skeletal muscle (n = 3; *, P = 0.011). (F-H) Error bars represent SD. *, P < 0.001.
Pol λ can tolerate an 8-oxo-dG(anti):dC base pair at the primer terminus.Figure 5(A) Substrates used for crystallization of the 8-oxo-dG:dC extension complex. The incoming dNTP (dUMPNPP) is shown in magenta. The primer terminal base (dC) and corresponding templating base (8-oxo-dG) are shown in cyan and orange, respectively. (B) Overview of the 8-oxo-dG:dC extension complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) and an incoming dUMPNPP (magenta). The primer terminal dC and 8-oxo-dG are shown in cyan and magenta, respectively. Key residues that form the primer terminal base pair binding pocket (N513, R517 and Glu529) and are involved in catalysis (D427, D429 and D490) are shown in black. The nucleotide binding metal (B) is shown in neon. (C) 8-oxo-dG (orange) establishes a Watson-Crick base pair with dC (cyan) at the primer terminus. Tyr505, Arg517, and Glu529 (purple) interact with the minor groove of the base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ). (D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, incoming dUMPNPP in magenta, and primer terminal dC in cyan. Expectedly, Mg2+ (neon) is occupying the metal B site. However, the metal A is absent in the structure, resulting in a non-catalytic position of the 3′-OH. This is consistent with other structures containing an incoming non-hydrolyzable analog.
Figure 5Effect of the lack of HR factors in spontaneous and MMS-induced mutagenesis. The frequency of spontaneous and MMS-induced forward mutagenesis at the CAN1 locus was determined in mid-log phase cell cultures before (spontaneous mutagenesis) and after treatment with 0.01% MMS for 4 hours (DNA damage-induced). Thus, the frequency of DNA damage-induced mutagenesis includes the mutants that arose spontaneously before MMS addition. The frequency of mutagenesis was determined at 0.01% MMS because at higher MMS concentrations the loss of viability of the rad mutants did not allow us to select for canavanine-resistant cells. Survival (%) after 4 hours in MMS relative to the wild type (taken as 100) is shown below the genotypes. The mean and SEM of 6 (wild type), 9 (rad54∆) and 3 (rest) independent fluctuation tests are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test (one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively). Only the statistical analysis of the effect of rad52∆, rad57∆ and rad51∆ on rad54∆-mediated mutagenesis is included for clarity. All mutants displayed statistically significant differences with the wild type and not with each other in the absence of MMS.
Figure 4A, Immunoblots of H358 and A549 cells treated with vehicle (DMSO), AZD 4547 (5 µM) and BI2536 (5 nM), alone or in combination for 24h. B, C, Immunoblots (B) and viability assay (C) of H358 and A549 cells preincubated overnight with SP600125 (2 µM) and SB203580 (1 µM) and subsequently treated with AZD 4547 (5 µM) and BI2536 (5 nM) for 24 hours. Data were shown as mean ± SD (n=3); ***P<0.001, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. D, E, Immunoblots (D) and viability assay (E) of H358 and A549 cells transfected with E2F1-specific or control siRNAs followed by treatment (48 h post transfection) with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536(5 nM) for additional 24 hours. Data were shown as mean ± SD (n=3). **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. F, H358 cells transfected with E2F1-specific or control siRNAs for 24h were subsequently treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or combination for 48h before apoptotic assay. Data were shown as mean ± SD (n=3). *P<0.05, **P<0.01 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.
Figure 4(A) Body length of individual nsun mutants throughout development (n = 44,7,7,7,8,8) in ~4 hour windows. L1-L4 refers to the larval stages, YA and Ad to young adult and adult, respectively. Data information: In (A), error bars indicate the 95% confidence interval of the median. (B) Size of mutant nsun strains at young adult and egg-laying stages. Approximately 100 synchronised young adults of each strain were measured. Images were processed using custom algorithms to recognize C. elegans and measure their cross-sectional area. (C Viable progeny counts of wild-type and nsun mutant strains at 20°C (C) Automatic counting was done using a Matlab script which processed plate images in real-time. Data information: In (C), data are presented as mean ± SEM, One-way ANOVA. D) Viable progeny counts of wild-type and noNSUN strains at 20°C and 25°C (D). Automatic counting was done using a Matlab script which processed plate images in real-time. Data information: In (D), data are presented as mean ± SEM, unpaired two-tailed Student's t-test.
The effects of CMA on degradation of endogenous Htt.Figure 8(A) The effects of knockdown and overexpression of LAMP-2A and Hsc70 on endogenous Htt and p62. PC12 cells were transiently transfected with LAMP-2A, Hsc70 plasmid, vector control or LAMP-2A and Hsc70 siRNA for 48 h. Lysates were subjected to Western blot analysis with antibodies against Htt and p62. Values are the mean±SE of three independent experiments. **P<0.01 (compared with endogenous Htt without treatment). (B) The PC12 cell viability following change of CMA. Following knockdown or overexpression of LAMP-2A and Hsc70 for 48 h, cell viability was analyzed with the MTT assay. Values were given as mean ± SD of three independent experiments.
Figure 7A. DCX nucleates MTs directly by stabilizing early tubulin oligomers (curved PFs). CDC mediates this activity as well as the initial stabilization of the nascent GTP/GDP.Pi-MT lattice. Then NDC joins and/or replaces CDC along the mature GDP-MT lattice. B. Summary of the sequential involvement of the two DC domains at different stages of MT assembly, characterized by different (bent or straight) tubulin conformations and different nucleotide states. Straight arrows indicate stabilization of tubulin-tubulin contacts. The colour-coded T shapes suggest DC domain-dependent inhibition of structural change: CDC binding between bent tubulin dimers may restrict longitudinal curvature of the assembly in addition to stabilizing the lateral and longitudinal contacts between tubulin dimers, and the subsequent CDC and NDC binding to MT lattice appears to lock tubulin in the lattice-specific straight conformation, preventing MT depolymerization associated with straight-to-bent tubulin transition. C. Summary of the DCX-MT binding area at different stages of MT maturation.
IL-2 pDHSs can be maintained in vitro by IL-7 in place of IL-2 A.Figure 7DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB IL‑2nil (data taken from Figure 2A), duplicate DNase-seq samples for TB IL-7 and TB IL-7nil are shown in parallel for the same sites. Average DNase-Seq tag density profile for the 1000 IL-2 pDHSs in both replicates of TB IL-7 and TB IL-7nil. UCSC genome browser tracks with DNase-Seq data for TB IL-7 and TB IL-7nil showing examples of IL-2 pDHSs within the Locus Control Region (LCR) within the Rad50 gene which controls the Th2 cytokines Il13, Il4 and Il5, plus IL-2 pDHSs within the Tnfrsf9 and Cish loci (C) IL-2 pDHSs are highlighted with blue boxes. UCSC genome browser tracks with DNase-Seq data for TB IL-7 and TB IL-7nil showing examples of IL-2 pDHSs within the Locus Control Region (LCR) within the non-primed control regions from the CD3 gene cluster and the Tbp locus (D). IL-2 pDHSs are highlighted with blue boxes. Average DHS signal at the 1015 IL-2 regulated inducible DHSs in TB+ IL-7 and TB+ IL-7nil. Relative mRNA expression levels in TB+ IL-7 and TB+ IL-7nil. The expression level in the TB+ IL-7 is set to 1. (n=3, error bars represent standard deviation, P-values were calculated using a paired t-test with *p<0.05, **p<0.005, ***p<0.0005.)
Figure 2DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB IL-2nil (32694) ordered by increasing fold change of tag count for TB IL-2 compared to TB IL-2nil. The sequence tag density is also shown in parallel at the same sites for the DNase-Seq analyses of CD4 naïve T cells (TN), TB IL-2 DMSO and TB IL-2 Ruxolitinib and ATAC samples from in vivo derived Th2 (Shih et al., 2016), Th1 and Th17 cells. The location of the 1000 IL-2 pDHSs is shown to the right. Average DNase-Seq tag density profile for the 1000 IL-2 pDHSs in both replicates of TB IL-2 and TB IL-2nil samples, and for naïve T cells (TN). Number of IL-2 pDHSs which are detected in in vivo purified CD4 T cell subsets (Chen et al., 2020, Garg et al., 2019, Shih et al., 2016). Overlap between the subsets of IL-2 pDHSs which are present in in vivo derived Th1, Th2 and Th17 cells. UCSC genome browser tracks with examples of IL-2 pDHSs within the Locus Control Region (LCR) within the Rad50 gene which controls the Th2 cytokines Il13, Il4 and Il5, plus IL-2 pDHSs within the Tnfrsf9 and Cish loci. IL-2 pDHSs are highlighted with blue boxes.
Figure 4A) Representative images of DIV4 primary hippocampal neurons from wild type, Ttll1-/- and Ttll7-/- mice Cells were incubated with MitoTracker® (black dots) and mitochondria movements were recorded in the longest neurite (orange lines). B) Kymographs of one representative experiment representing mitochondrial movements C - F) Analysis of one single experiment shown in (B). Scatter dot plots of (C) run length and (D) speed distributions, where each point represents a single run extracted from the kymographs shown in (B). The black lines indicate the median of the distribution, with whiskers at interquartile range. Numerical values of medians are indicated below each plot. (E) A bar graph showing the overall motility of mitochondria (the fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s). (F) A bar graph showing mitochondrial density in analysed axons. Values of the overall motilities and mitochondrial densities are given below the bar plots. G - J) Statistical analyses of mitochondrial transport parameters of five independent experiments shown in (D-G and Fig. EV3A-D). Each bar represents the mean (±SEM) of medians of single experiments for (G) run length and (H) speed distributions, and average (±SEM) (I) overall mitochondrial motility and (J) density of all five experiments. Values of fold change between control and Ttll1‑/- and Ttll7-/- conditions are indicated below the graphs. Full bars are for anterograde, and empty bars for retrograde transport events. Significance was tested using unpaired t-test. Average (G) run length as well as (H) speed showed no significant differences between control and Ttll1-/- and Ttll7-/- conditions. (I) The averages of overall motilities reveal an about 1.7-fold increase in overall mitochondrial motility in Ttll1-/- neurons, while no significant differences were found in Ttll7-/- neurons. (J) Mitochondrial density was unaltered between control and knockout conditions. Total number of mitochondria analysed from five independent experiments: control (1260), Ttll1-/- (1158) and Ttll7-/- (1191).
Cycloheximide blocked sodium butyrate induced endoplasmic reticulum stress in colorectal cancer cells.Fig 6  HCT-116 or HT-29 cells were treated with 10 μg/mL cycloheximide for 30 min followed by sodium butyrate (NaB) for 24 h. Representative Western blots showed the expression of IRE-1a, BIP, PDI, and GADPH (loading control) in HCT-116 (A) or HT-29 (C) cells. Protein expression was quantified by densitometry and normalized to GAPDH (ratio of protein:GAPDH). The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT-116 (B) and HT-29 (D) cells. are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group.
A, B) Two-dimensional tryptic mapping of YFP–p27, YFP–p27T198A and a mixture of both (A).figf1(A, B) Two-dimensional tryptic mapping of YFP-p27, YFP-p27T198A and a mixture of both (A). Phosphoamino-acid analysis of spots a and b (B). The migration of phosphoserine (S), phosphothreonine (T) and phosphotyrosine (Y) is marked with circles in B. The asterisk indicates the origin of migration. (C-E) MCF-7 transfected with YFP-p27 or YFP-p27T198A mutant (C, D) or cotransfected with YFP-p27 and an empty RFP vector in a 5:1 (YFP-p27: RFP) ratio (E) were treated with (+) or without (−) MG132 (10 μM) for 12 h before western blot analysis (C) and fluorescence microscopy (D, E). (C-E) MCF-7 transfected with YFP-p27 or YFP-p27T198A mutant (C, D) or cotransfected with YFP-p27 and an empty RFP vector in a 5:1 (YFP-p27: RFP) ratio (E) were treated with (+) or without (−) MG132 (10 μM) for 12 h before western blot analysis (C) and fluorescence microscopy (D, E). (F) Western blots of protein extracts from MCF-7 transfected with YFP-p27, YFP-p27T198A or YFP-p27T198D mutants following incubation with cycloheximide (CHX) for the indicated times. Uncropped images of the blots are shown in the Supplementary Information, Fig. S5.
Figure 5(a) The amount of glycogen in the whole larvae measured by biochemical assay (n=4), normalized to RasV12; ctrl at day 6, and per number of larvae. (b-e) Representative confocal images of muscle and adipose tissue of larvae carrying RasV12; ctrl, RasV12; scrib-/- and RasV12; scrib-/-, atg13-/-//atg13-/- showing glycogen levels (white) at day 6 and day 8. (f) Cartoon illustrating tumor growth (green) incorporation of molecules derived from food (in orange) or from host tissues (in blue). (g) Sources of carbon incorporated into tumor biomass were differentiated by changing the isotopic carbon content of the food source 25 hours before measuring the total carbon content and isotopic ratio of the tumor. A similar, but independent experiment is reported in Holland P., et al., 2021 BMC Biol.
Fig.Figure 4A-C Squalene (A), lanosterol (B) and cholesterol (C) abundance in p53+/+ and p53-/- HepG2 cells transfected with control or SQLE siRNA for 48 hours were determined via ultra-high pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS). Protein levels were analyzed by western blotting with specific antibodies (A bottom panel). D p53+/+ and p53 -/- HCT116 cells were transfected with control or SQLE siRNA for 48 hours as indicated. Relative cholesterol concentrations were examined as described in methods. Protein expression were determined by western blot. E and F Cholesterol levels of HCT116 cells (E) and HepG2 cells (F) treated with or without 30μm terbinafine for 72 hours. G and H Triglyceride levels in p53+/+ and p53-/- HCT116 cells (G) and HepG2 cells (H) treated with SQLE siRNA or control siRNA for 48 hours.
Figure 5A. Analysis of proOmpA:SecB and TF:proOmpA:SecB complexes by native-MS. SecB4 (30 μM; Buffer G), SecB4 (40 μM) and proOmpA (20 μM; pre-treated with 10 mM DTT; buffer G) and of SecB4 (40 μM), proOmpA (20 μM) and TF2 (20 μM) in buffer G were analyzed as in Fig. 2B (raw data in Fig. EV3). n=2 biological replicates. B. Blow up of the ESI-mass spectrum of the TF:proOmpA:SecB4 super-complex from A (complete dataset in Fig. EV3). Left: charge state distributions, at the indicated m/z range. Right: masses deconvoluted from the mass spectra (as in Fig. 2B). Inset cartoon depicts stoichiometry. Representative spectrum shown; n=2 biological replicates. C. TF:proOmpA:SecB supercomplex analysis using GPC-MALS in Buffer A. TF1:proOmpA1 (10 µM each; green), SecB4 (40 µM; blue) and TF1:proOmpA1:SecB4 (10, 10 and 40 µM, respectively; black). n= 5 biological replicates; mean mass: 137 kDa (± 6 kDa) representative experiment shown; red circles: determined mass (kDa); AU: arbitrary units.
Figure 1Genome organization of SARS-CoV and schematic illustration of Nsp3 domains. Ubl1: ubiquitin-like domain 1; Mac: macrodomain; DPUP: Domain Preceding Ubl2 and PLpro; PLpro: papain-like protease; NAB: nucleic-acid binding domain. SUD binding to Paip1 in HEK-293 cells as demonstrated by the split-YFP assay. Schematic presentation of SUD, Paip1, and PABP. Interactions between SUD and Paip1, as well as PAPB and Paip1 are indicated by red and black lines, respectively. PAM: PABP-binding motif; Paip1M: the middle domain of Paip1; RRM: RNA-recognition motif; PABC: the C-terminal domain of PABP. Paip1M alone does interact with SUD. SUD binds to Paip1 and Paip1M with the same binding intensity. n. s.: not significant. The Mac2 subdomain is crucial for interacting with Paip1M. Paip1M and SUD interaction was confirmed in vitro by a gel-filtration assay.
TNF-α responses to A.Figure 9Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage. Cells were pre-treated for 1 h with FK506 (10 ng/ml), the NFAT inhibitor MCV1 (1 μM) or SC514 (10 μM) before stimulation with A. fumigatus swollen conidia (MOI 0.1). After 24 h of stimulation, TNF-α was measured in the cell culture supernatant by ELISA.A Representative FACS-phenotyping demonstrative of a normal alveolar macrophage phenotype is shown for three patient samples. Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage. Cells were pre-treated for 1 h with FK506 (10 ng/ml), the NFAT inhibitor MCV1 (1 μM) or SC514 (10 μM) before stimulation with A. fumigatus swollen conidia (MOI 0.1). After 24 h of stimulation, TNF-α was measured in the cell culture supernatant by ELISA.B Inhibition of alveolar macrophage responses to AF with FK506, MCV1 or SC514 led to a significant reduction in TNF-α levels.