EMBO/sd-character-level-ner huggingface dataset:
ER stress and UPR response upon Q depletion A Transmission electron microscopy images showing the increase of cystic ER vacuolae upon q depletion.Figure 6A Transmission electron microscopy images showing the increase of cystic ER vacuolae upon q depletion. Arrowheads indicate rough endoplasmic reticulum while expansions are indicated by asterisks. Scale bar 250 nm. Data information: S (standard medium), SF (serum free medium), q (queuine). B Representative images showing immunofluorescence of HeLa cells grown in S and SF medium in the absence or presence of Q. Anti-KDEL (ER stress marker); anti-eIF2-pS51 (ER stress-induced inhibitor of translation); anti-ATF4 (transcription factor that activates ER stress-responsive genes); anti-Hps70 (chaperone). Nuclei were stained with Hoechst. Scale bar 10 µm. Data information: S (standard medium), SF (serum free medium), q (queuine).
Figure 1Scheme of the cytoplasmic tRNA thiolation pathway. Nfs1 mobilizes the sulfur and transfers it via a persulfide onto the rhodanese-like domains (RHDs) of either Tum1 or Uba4. Uba4 catalyzes the activation and thiocarboxylation of Urm1. Urm1-COSH is used by the Ncs2/Ncs6 complex to form 2-thiouridine on tRNAs. AD: adenylation domain. RHD: Rhodanese-like domain Domain organization of CtUba4. AD: adenylation domain. RHD: Rhodanese-like domain Size exclusion chromatography profiles of recombinant proteins used in this study. AU: arbitrary units; WT: wild type; FL: full-length. Cartoon representation of the CtUba4 dimer. The AD dimer is emphasized by a transparent surface representation and disordered loops are shown as dashed lines. The AD and RHD active loops are colored in red and purple, respectively. Zn atoms are shown as grey spheres. The two interwound Uba4 monomers are shown in black and white (central inset). Structural comparison of CtUba4 AD with EcMoeB (PDB ID: 1JW9) and of CtUba4 RHD with HsMOCS3 RHD (PDB ID: 3I2V). r.m.s.d. values as a measure of structural similarity are indicated.
Figure 4A Immunoblotting of protein extracts from 1205Lu cells transfected with a siRNA control (CTRL) or two different sequences of siRNA (#1 and #2) directed against DDR1 or DDR2 alone or in combination prior being cultivated on MAF-derived ECM and treated or not with 5 µM BRAFi for 96 h, using antibodies against the indicated proteins. HSP60, loading control. B Immunoblotting of protein extracts from SKMEL5 cells transfected with siCTRL or the combination siDDR1#2/siDDR2#2 prior being cultivated on FRC- or MAF-derived ECMs (left and right panels, respectively) and treated with vehicle, 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi, using antibodies against the indicated proteins. HSP60, loading control. C Quantification of the time-lapse imaging of the proliferation of NucLight-labeled 1205Lu cells using the IncuCyte ZOOM system. Cells were plated for 48 h on FRC- or MAF-derived ECMs prior a 96 h treatment with vehicle or 5 µM BRAFi in the presence or not of 7 µM Imatinib or 1 µM DDR1-IN-1. The bar plots represent the mean normalized to vehicle of NucLight red nuclear objects per field ± SEM from 3 independent experiments performed in triplicate. ****P<0.0001, two-way ANOVA followed by Sidak's multiple comparison test. D Immunoblotting of protein extracts from 1205Lu, SKMEL5 and MM099 cells plated for 48 h on FRC-derived ECM prior a 96 h treatment with vehicle or 5 µM BRAFi in the presence or not of Imatinib (7 µM for 1205Lu, 10 µM for SKMEL5 and MM099) or DDR1-IN-1 (1 µM for 1205Lu, 5 µM for SKMEL5, 3 µM for MM099), using antibodies against the indicated proteins. HSP60, loading control. E Flow cytometry analysis of cell death (Annexin V/PI labeling) in 1205Lu cells plated on FRC-derived ECM and treated as above. Right bar plots show the distribution of cells (% of total) across the different forms of death.
a, Loss of nuclear REST in DLB and FTD.f5a, Loss of nuclear REST in DLB and FTD. Left, immunofluorescence microscopy with labelling for REST (green), the neuronal marker MAP2 (red) and DNA (DAPI, blue). Right, quantitative analysis of immunofluorescence. Values (AFU) represent the mean ± s.e.m.; Young, n = 11; Aged, n = 21; DLB, n = 18; FTD, n = 16. **P 0.001 by Student's unpaired t-test. b, Colocalization of REST with disease-associated misfolded proteins in autophagosomes. Cortical sections were triple-labelled for REST, LC3, and either Aβ in AD, α-synuclein (α-syn) in DLB, or phosphorylated tau (PHF1 epitope) or TDP-43 in FTD. Two different representative cases are shown for each disease. Scale bars, 20 µm.
Figure 2A Confocal microscopy of U2OS cells ectopically expressing TERT-WT or TERT-K626A. Total dsRNA is stained green, and nuclei are stained blue (DAPI). Scale bar = 20 µm. B Genome-wide upregulation of ERVs in U2OS cells ectopically expressing TERT-WT or TERT-K626A. RNA-seq data for TERT-WT/TERT-K626A and empty vector control were used to calculate the log2 fold change values. Red dots indicate elements with increased expression, while blue dots indicate the elements with decreased expression. C Representative TA-ERVs activated by TERT-WT or TERT-K626A. D RT-qPCR analysis of TA-ERVs in U2OS and HeLa cells ectopically expressing TERT-WT or TERT-K626A. E RT-qPCR analysis of TA-ERVs in HeLa and HCT116 cells with TERT knockdown. F F-H RT-qPCR analysis of interferon-related genes and TA-ERVs (F), western blot analysis of TERT, TBK1, pTBK1, IRF3, pIRF3, and GAPDH levels (G), and ELISA of IFNβ and CXCL10 in culture supernatant (H) in U2OS cells ectopically expressing TERT-WT or TERT-K626A using Dox-inducible system. GFP was used as negative control. Relative levels of pTBK1 and pIRF3 were quantified with ImageJ software and normalized to GAPDH, as indicated at the bottom of the blot (G).
Figure 1(A-B) Human fibroblasts (BJ) were treated with vehicle (water for 8 times 24 hours) or abemaciclib (1 μM for 8 times 24 hours). The population doubling over the 8 days treatment is plotted (A). 1 or 8 dpt, cells were incubated with EdU for 20 hours and stained and quantified (n=9 samples from 3 independent experiments; scale bar, 150 μm) (B). Data information: Data are means ±SD. Unpaired student t-test *p<0.05, **p<0.01, ***p<0.001. dpt, days post treatment. (C) Chromatin was extracted from BJ cells treated with vehicle or abemaciclib (1 μM for 8 times 24 hours) or nutlin-3a (10 μM for 8 times 24 hours) or doxorubicin (250 nM for 24 hours) and ChIP assays using an antibody against p53 were performed. qRT-PCR was performed using primers amplifying promoter regions of CDKN1A, GADD45A and MDM2 genes containing p53 binding sites. Values indicate fold enrichment relative to the vehicle group (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001. (D) BJ cells transduced with a p53 reporter were treated with nutlin-3a (10 μM, positive control) or abemaciclib (1 μM for 1 or 4 or 8 times 24 hours) and luciferase activity measured after treatments at the indicated time points (n=3 independent experiments). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001. (E) 3*103 BJ cells of the indicated genotypes were re-plated after vehicle or abemaciclib treatment (1 μM) and stained with 0.2% crystal violet 8 dpt (n=3 independent experiments). (F) 8 dpt, treated BJ cells were incubated with EdU for 20 hours and stained (n=9 samples from 3 independent experiments). ( Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001. (G) 3*103 mouse dermal fibroblasts (MDFs) of the indicated genotypes were re-plated after vehicle or abemaciclib treatment (4 μM for 8 times 24 hours) and stained with 0.2% crystal violet 8 dpt (n=3 independent experiments). (H) 8 dpt, treated MDFs were incubated with EdU for 20 hours and stained (n=9 samples from 3 independent experiments). ( Data information: Data are means ±SD. Unpaired student t-test *p<0.05, **p<0.01, ***p<0.001. (I) RNA was isolated from vehicle or abemaciclib (1 μM) treated scramble/shp53 BJ cells and mRNA for the indicated genes quantified by qRT-PCR relative to tubulin (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.
Fig.Figure 3(A) Schematic of network analysis to identify TFs with activity (increased or decreased) in controlling the transcriptional state of MTB during infection of host cells. Abbreviations: dec; decreased, inc; increased. (B) Heatmap of TFs with decreased (purple) or increased (green) activity at specific time points during in vitro or in vivo infection. (C) Rv0681 regulon genes differentially expressed in vitro and the evidence for predicted high Rv0681 activity (induced expression of target genes). (D) Zur regulon genes differentially expressed in vitro and the evidence for decreased Zur activity (de-represses target genes). (E) Rv0691c regulon genes differentially expressed in vitro and in vivo; evidence for increased Rv0691c activity (increased up- and down-regulation of target genes) at 24 h from both in vitro and in vivo infection.
The expressions of H19 and Igf1r are significantly decreased in the eutopic endometrium of women with endometriosis compared to those without endometriosis.Figure 1A-F (A, B, D-F) RNA levels determined by RT-qPCR. Results are presented as mean ± SD,n = 10. P-values are indicated on the top of each plot. (C) Spearman correlation suggests an in vivo positive correlation between the expressions of H19 and Igf1r in a statistically significant manner. Spearman correlation coefficient, P-values, and sample numbers are marked on the top left of the plot.
Figure 4(A) Western blot analysis of putative exosome markers in lysates from HCT116 cells cultured in glutamine-replete (2.00 mM) and glutamine-depleted (0.15 mM) medium for 24 h. Gel was loaded with equal amounts of protein (total cell lysate protein was reduced by 19 ± 4 % after glutamine depletion). The activity of mTORC1 was assessed via phosphorylation of S6 and 4E-BP1, using phospho-specific antibodies and a pan-4E-BP1 antibody, where the hyper-phosphorylated form of 4E-BP1 produced by mTORC1 gives the slowest migrating γ band. Bar chart shows the abundance of putative exosome proteins relative to tubulin in these lysates. Western blot analyses of EV preparations. (B) EVs isolated by ultracentrifugation (UC) of medium from HCT116 cells cultured in glutamine-replete and -depleted conditions for 24 h, and loaded according to cell lysate protein levels to compare secretion on a per cell basis, as shown in bar chart from four biological replicates. Western blot analyses of EV preparations. (C) EV preparation isolated by size-exclusion chromatography (SEC) from glutamine-depleted HCT116 cells and then separated by high-resolution iodixanol-PBS density centrifugation. Note that exosomes (exos) and microvesicles (MVs) are found in only partially overlapping fractions and the pattern of Rab11a separation mirrors that of exosome markers (primarily 1.064 - 1.085 g/ml). Western blot analyses of EV preparations. (D) EVs isolated by UC under glutamine-depleted conditions as in (B) and then subjected to Proteinase K (Prot K) digestion in the presence or absence of Triton® X-100 (Triton-X). Only membrane-associated CD81 is digested in the absence of Triton® X-100, while CD63 is resistant to digestion, even in the presence of Triton® X-100. Western blot analyses of EV preparations. EVs isolated under glutamine-depleted conditions were immunocaptured with anti-CD63 antibodies coupled to magnetic beads. This method only pulls down a fraction of CD63, so the protein from pull-down of eight times the input is loaded for comparison. Approximately the same low levels of Rab11a are captured by control IgG and anti-CD63 beads. Western blot analyses of EV preparations. (F) EVs isolated under glutamine-depleted conditions were immunocaptured with anti-CD81 antibodies coupled to magnetic beads. All or most CD63 and CD81 appears to be pulled down, but less than 10% of Rab11a and Cav-1 is captured. NTA analysis suggests about 10% of all particles are pulled down with this approach.
Figure 3A Schematic illustration of developmentally expressed olfactory cell types: horizontal basal cell (HBC), globose basal cell (GBC), intermediate nerve progenitor (INP), and immature olfactory sensory neuron (iOSN), mature olfactory sensory neuron (mOSN) and sustentacular cell (Sus). B The mRNA levels of CK14, Trp63, Sox2 and Mash1 in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; CK14: p=0.2135, Trp63: p=0.1930, Sox2: p=0.5190, *p<0.05; Student's t-test). C Representative IF staining for CK14, Sox2, and Mash1 in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. D Quantification of the number of CK14+, basal Sox2+, apical Sox2+, and Mash1+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; CK14: p=0.8998, basal Sox2: p=0.7056, apical Sox2: p=0.4455, **p<0.01; Student's t-test). E Western Blot analysis of the Sox2 and Mash1 protein levels in the MOE of the NC and miR-200b/a KD mice (n=2 mice each group). GAPDH and Tubulin served as loading controls. The molecular weight of each band is indicated at the right. F Representative IF costaining with Sox2 antibody and EdU in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both Sox2 and EdU. F' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (right images). F: Scale bars, 20 μm; F': Scale bars, 2 μm. G Quantification of the number of Sox2+EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; p=0.7988; Student's t-test). H Representative IF costaining with Mash1 antibody and EdU in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both Mash1 and EdU. H' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (right images). H: Scale bars, 20 μm; H': Scale bars, 2 μm. I Quantification of the number of Mash1+EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test).
Figure 1(A) Representative Opera images of WT and CLN3 KO ARPE 19 cell lines stained using fluorescent-conjugated cholera toxin (to detect GM1), LipidTox (to detect neutral lipids), fluorescent-conjugated Shiga toxin and is quantification (to detect Gb3), Filipin III (to detect cholesterol). Data are presented as mean ± SD ***: P ≤ 0.0001, as determined by Student's t‐test (n=3 biological replicas in duplicate). Scale bars: 50 µm. (B-C) Representative confocal images and their quantification of Gb3 accumulation within the lysosome, detected by fluorescent-conjugated Shiga toxin, from WT, CLN3-KO and CLN7-KO ARPE-19 cells treated with PDMP or silenced for Gb3 synthase (siGb3S) (*** vs WT, °°° vs DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm. (D) Lipidomic analysis of ARPE-19CLN3KO and ARPE-19CLN7KO. Data are presented as mean ± SD, *: P ≤ 0.01, **: P ≤ 0.001, ***: P ≤ 0.0001, as determined by ANOVA (n=4 technical replicas).
．Interaction of Bax (α2-α5) with lipid bicelles NOE strips taken from 3D 15N-edited NOESY-TROSY-HSQC spectrum (200 ms NOE mixing time) recorded at 900 MHz using the [15N, 2H]-labeled Bax (α2-α5) in DMPC/DHPC bicelles with q = 0.Figure 3NOE strips taken from 3D 15N-edited NOESY-TROSY-HSQC spectrum (200 ms NOE mixing time) recorded at 900 MHz using the [15N, 2H]-labeled Bax (α2-α5) in DMPC/DHPC bicelles with q = 0.55. The indicated crosspeaks in the aliphatic regions are the NOEs between the protein backbone amide protons and the lipid aliphatic protons. Residue-specific PREamp of Bax (α2-α5) in DMPC/DHPC bicelles with q = 0.6 determined from the lipophilic PRE analysis. The PREamp values were derived from the 16-DSA titration. The plot is colored according to the PREamp values that are proportional to the effects of 16-DSA on the individual residues of Bax (α2-α5). The residues with the least PREamp values in the blue colored areas are in the flexible terminal regions. Schematics of a Bax (α2-α5) dimer structure in ribbon representation relative to a bicelle with DMPC (blue) in the lipid bilayer and DHPC (pale yellow) in the rim. The protein structure contacts the DMPC lipid bilayer directly with a 60° tilt angle, which is illustrate more clearly by the schematic (D). Position of a Bax (α2-α5) dimer structure in ribbon representation relative to the lipid bilayer of a bicelle. The protein structure is placed against the lipid bilayer with a 60° tilt angle between its longest axis (L) and the bilayer normal axis (N) as derived from the best sigmoidal fitting. The hydrophobic α4-α5 surface contacts the lipid bilayer. Amide protons are shown as spheres color-coded similarly as in panel B. The polar headgroup region and the nonpolar acyl tail region of the lipid bilayer are indicated by light blue and light orange colored strips, respectively. The estimated thickness for the DMPC lipid bilayer is indicated on the left side. The Bax (α2-α5) dimer structure on the left is rotated 180° to generate the structure on the right showing the nonpolar residues in the α4-α5 surface, most of which contact the nonpolar core of the lipid bilayer. Note that the two positively charged R89 residues on the boundary of the nonpolar surface are close to the polar lipid headgroups.
Figure 8qRT-PCR detection of Ifnb1 mRNA (A) of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are means+SD averaged from 3 independent experiments performed with technical triplicates. Each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001. ELISA detection of IFNβ in supernatants (B) of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are means+SD averaged from 3 independent experiments performed with technical triplicates. Each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001. Western blot detection of indicated proteins in lysates of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are representative of 3 independent experiments.
Figure 3(A-C) LPS-primed BMDMs were pre-treated with the indicated dose of LicoB for 1 h and then stimulated with ATP for 1 h. Western blot analysis was used to detect cross-linked ASC in the Triton X-insoluble pellet, the arrow indicates ASC (A). Caspase-1 activity (B) and IL-1β secretion (C) in the SN were measured again to verify NLRP3 activation. (D) Western blot analysis of cross-linked ASC in the Triton X-insoluble pellet from LPS-primed BMDMs pre-treated with LicoB (20 μM) or vehicle and then stimulated with nigericin, ATP, poly(I:C), and MSU. Pam3CSK4-primed BMDMs were treated with LicoB (20 μM) and then stimulated with LPS. (E) Western blot analysis of cross-linked ASC in the Triton X-insoluble pellet from LPS-primed BMDMs pre-treated with LicoB (20 μM) or vehicle and then stimulated with nigericin, poly (dA:dT), or Salmonella.
Specific binding of RK-33 to DDX3 and induction of radiosensitization in lung cancer cell lines.Figure 2A Chemical structure of RK-33.B A predicted molecular model of RK-33 docked into the ATP-binding cleft of DDX3. RK-33 is displayed in pink, the surface of the DEADc domain is in green, and the surface of the HELICc domain is in red.C Hydrogen bond interactions between RK-33 and DDX3. Alpha helices are displayed in green, and β-sheets are shown in maroon. D, E Chemical structures of biotin-linked RK-33 at R3 position with ethylene amine linker (AK-298) and biotin-labeled RK-33 at R2 position with (PEG) 2 ethylene amide linker (SK-153). The two structural differences of AK-298 and SK-153 are the length of the biotin linker and attachment position at RK-33. F Immunoblots of pull-down assay of DDX3 with biotin, AK-298, and SK-153. Lower panels display results using DDX5 and DDX17 antibodies. G Schematic representation of helicase assay. H Immunoblot displaying increasing concentrations of RK-33 (50, 100, 200 nM) resulting in increased inhibition of unwinding of oligomer products (lanes 4-6). I MTS viability assay of various lung cancer cell lines treated with RK-33 for 72 h. Mean from 3 replicates with SD. J, K Colony-forming assay of A549 and H3255 cells treated with RK-33 and with various doses of radiation 4 h later. Curves were fitted with a quadratic polynomial equation. Mean from 2 replicates with SD. P-values were determined by the extra sum of squares F-test.
Ref(2)P localizes to protein aggregates in flies that have impaired autophagy or proteasome function.Figure 3.(A and B) atg8a mutants exhibiting a striking positive staining for Ref(2) P and ubiquitin staining in all the regions of the adult brain. As it is shown in high magnification of the optic lobe (medulla region; B), Ref(2) P- and ubiquitin-positive aggregates are observed (arrows). Genotypes: (A and B) atg8aKG07569/Y (C) Normal phenotype is observed in β2 and 6 dominant-negative temperature-sensitive mutants at 25°C. (D) Accumulation of Ref(2) P- and ubiquitin-positive aggregates are observed in β2 and 6 mutants at 29°C (arrows). (C and D) elavGal4/+; UAS-β6/+; UAS-β2/+ (E and F) double mutants for atg8a and ref(2) P loss-of-function alleles lacking the PB1 (E) or UBA (F) domain do not form ubiquitinated protein aggregates. Ubiquitin exhibits a cytoplasmic staining pattern (arrows). Genotypes: (E) atg8aKG07569/Y; ref(2) Pod2/ref(2) Pod2, and (F) atg8aKG07569/Y; ref(2) Pod3/ref(2) Pod3.