EMBO/sd-character-level-ner huggingface dataset:
Figure 6Western blot image (above) and quantification (bottom) of TRF1 protein levels upon genetic depletion of ERK1/2 in p53-/- MEF line. Data are representative of n=3 independent experiments. Representative images (above) and quantification (bottom) of TRF1 telomeric foci in ERK1/2 RNA interfered p53-/- MEFs. Scale bars, 5μm. Data are representative of n=2 independent experiments. p53-/- MEFs were sequentially transduced with lentiparticles encoding short hairpins against ERK1/2 and retroparticles for eGFP-tagged Trf1 WT or mutant alleles to overexpress TRF1 depicted variants in the absence of ERK1/2. Western blot image of eGFP-tagged and endogenous TRF1 protein levels upon genetic depletion of ERK1/2 in p53-/- MEF line. Representative images (above) and percentage (bottom) of telomeric and 53BP1 colocalizing foci (TIFs) per cell in p53-/- MEF with or without overexpression of eGFP-Trf1 WT and indicated mutants upon genetic depletion of ERK1/2. White arrowheads: colocalization of telomeric and 53BP1.Scale bars, 10μm.
Role of ABCE1 in eukaryotic translation initiation.Figure 8Schematic representation of eukaryotic translation initiation. ABCE1 is shown in orange, nucleotide states are symbolized with "T" for ATP-bound and "D" for ADP-bound; the two protomers of eIF3j are shown in blue and green, the eIF3j C-terminus of protomer 2 in mRNA entry channel is marked with an asterisk.
Figure 1Representative immunofluorescence of primary cilium in WT or Mecp2 null MEF cells. Cells were starved for 48 h before staining with anti-γ-tubulin (green) and anti-acetylated α-tubulin (red) antibodies. Merge of all channels and DAPI staining (blue) is depicted for both genotypes. Scale bar 20 µm, and 10 µm in the enlarged image. Arrows indicate not ciliated cells. Cilia were counted and analysed with ImageJ to quantify the percentage of ciliated cells (n=134 WT and n=140 Mecp2 null cells) (B) and the cilium length (n=104 WT and n=43 Mecp2 null cells) (C). The graphs show the average of three independent experiments (mean ± S.E., **p<0.01, by Mann-Whitney test; ***p<0.001, by Student's t-test). Representative immunofluorescence of primary cilium in hTERT-RPE-1 cells silenced with MECP2- or control-siRNAs. Cells were starved for 48 h before staining with anti-γ-tubulin (green) and anti-acetylated α-tubulin (red) antibodies. Scale bar 10 µm, and 5 µm in the enlarged image. Arrows indicate not ciliated cells. Representative Western blotting showing the MeCP2 expression in CTRL and silenced hTERT-RPE-1 cells, with respect to GAPDH, used as loading control. The percentage of MeCP2 positive cells was calculated by analysing n=1000 CTRL and silenced hTERT-RPE-1 cells (mean ± S.E., ***p<0.001, by Student's t test). Cilia were counted and analysed with ImageJ to quantify the percentage of ciliated cells (n=191 CTRL and n=197 siMeCP2 cells) (G) and the cilium length (n=50 CTRL and n=50 siMeCP2 cells) (H). The graphs show the mean ± S.E of three independent experiments (***p<0.001, by Mann-Whitney test (G) or Student's t-test (H)). Representative immunofluorescence of primary cilium in WT and Mecp2 null primary cortical neurons at DIV7 and DIV14. Scale bar 10 µm, and 5 µm in the enlarged image. Arrows indicate not ciliated cells. Neurons were fixed at DIV3, DIV7 and DIV14 and primary cilium detected by immunostaining with anti-AC3 antibody. Percentage of ciliated cells (J) and cilium length (K) were analysed. Histograms represent the average of three independent experiments, analysing neurons derived from 3 different experiments (n=156 WT DIV3; n=49 KO DIV3; n=177 WT DIV7; n=82 KO DIV7; n=59 WT DIV14; n=37 KO DIV14) (mean ± S.E., **p<0.01, ***p<0.001, by two-way ANOVA followed by Bonferroni post hoc test). Primary cortical astrocytes were fixed at DIV15 and stained with anti-acetylated α-tubulin (red) and anti-ɣ-tubulin (green) antibodies. Scale bar 20 µm, and 10 µm in the enlarged image. Arrow indicates not ciliated cell. Percentage of ciliated cells (M) and cilium length (N) were analysed (n=26 WT and n=34 Mecp2 null astrocytes). Histograms represent the average of three independent experiments (mean ± S.E., *p<0.05, Student's t test).
Figure 3A Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple (red). The cells were treated with DMSO (Upper) or 5 μM Tg (Middle) for 30 min. After Tg treatment, the cells were washed with PBS and then returned to culture medium for 60 min (Lower). GM130 was immunostained (blue) as a cis-Golgi marker. Scale bar = 10 μm. B Pearson correlation coefficient calculated Data were shown as mean ± SEM of N > 80 cells from four independent experiments. ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test). C Detection of pS357-PDI and UPR signaling in HepG2 cells after Tg treatment and washout for indicated times. D HepG2 cells were treated with 25 ng/μl CHX for indicated times and analyzed by immunoblotting for pS357-PDI and UPR signaling. 5 μM Tg or DMSO was added 30 min before cell harvest. E Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple (red) pretreated with 25 ng/μl CHX for 7.5 h, followed by introduction of DMSO (Upper) or 5 μM Tg (Lower) for 30 min, respectively. GM130 was immunostained (blue) as a cis-Golgi marker. Scale bar = 10 μm. F Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple-KDEL (red) treated with DMSO (Upper) or 5 μM Tg (Lower) for 30 min, respectively. Scale bar = 10 μm. G HepG2 cells were transfected with empty vector (-), FLAG-tagged Fam20C or Fam20C-KDEL followed by introduction without or with 5 μM Tg for 30 min, respectively. PDI phosphorylation was detected by protein immunoblotting on both regular and phostag gels. p-PDI percentage was quantified based on band intensities on phostag gels.
Figure 5(A) The interaction of EPCR1 with HAM1 and HAM2. EPCR1-Flag transgenic plants were crossed to HAM1-Myc and HAM2-Myc transgenic plants. The progeny were used to evaluate the interaction between EPCR1 with HAM1 and HAM2 by co-IP. (B) The interaction of ARID2, ARID3, and ARID4 with HAM1 and HAM2. ARID2-Myc, ARID3-Myc, and ARID4-Myc transgenic plants were crossed to HAM1-Flag and HAM2-Flag transgenic plants and their progeny were used for co-IP. (C) The interaction of TRB1 with HAM1 and HAM2. TRB1-Flag transgenic plants were crossed to HAM1-Myc and HAM2-Myc transgenic plants and their progeny were used for co-IP. (D) The interaction of HDA9 with ARID2, ARID3, and ARID4. ARID2-Myc, ARID3-Myc, and ARID4-Myc transgenic plants were crossed to HDA9-Flag transgenic plants and their progeny were used for co-IP. (E) The interaction of HDA6 with EPCR1 and ARID2. EPCR1-Flag and ARID2-Flag transgenic plants were crossed to HDA6-Myc transgenic plants and their progeny were used for co-IP.
Figure 6A. Silver-stained SDS-PAGE of Elongator glycerol gradient fractions purified using low salt (75 mM NaCl) buffer. The fraction containing the most Elp4, Elp5, and Elp6 is designated by an asterisk (*) and the corresponding fraction from a glutaraldehyde-containing glycerol gradient is used for EM analysis. B. Comparison of class averages of Elongator purified in buffer containing regular or low salt concentrations (150 mM and 75 mM NaCl, respectively). Densities resembling a second copy of Elp456 is indicated by white triangles. C. Scheme of the Elongator and rElp456 mixing experiment. Recombinant Elp456 subcomplex (rElp456) was purified from E. coli and mixed with native yeast Elongator in regular salt conditions. The mixture, as well as rElp456 alone, were then subjected to glycerol gradient ultracentrifugation. D. Coomassie-stained SDS-PAGE of rElp456 glycerol gradient fractions. E. Coomassie-stained SDS-PAGE of the Elongator mixed with rElp456 glycerol gradient fractions. The fraction containing the most intact complexes is indicated with an asterisk (*), and the corresponding fraction from a glutaraldehyde-containing glycerol is used for EM analysis. F. Comparison of class averages of natively purified Elongator with and without rElp456 incubation. Densities resembling a second copy of Elp456 are indicated by white triangles.
Figure 4A t-SNE plots showing the original (left) and re-clustered (right) osteogenic lineage cells by integrated analysis of homeostatic and stress conditions. Top 10 PCs were chosen for the clustering. B Pseudotime analysis by Monocle 2. Arrow indicated inferred direction of the differentiation trajectory. C Heatmap showing pseudotime-dependent gene expression. Bars on the top were colored as in (A). Representative genes were highlighted on both sides. D Frequencies of different osteogenic clusters under homeostatic and stress conditions. E Heatmap showing the AUC score of regulons enriched in each stress. Z-score (row scaling) was calculated. Representative regulons were highlighted on the right. F Representative images of adipogenic, osteogenic and chondrogenic differentiation following shRNA knockdown in Lepr-Cre+ BMSCs. Scale bars were 100 µm. G qPCR analysis of the expression levels of Pparg, Bglap and Acan in control or knockdown group (n=3 independent experiments). The statistical significance of differences was analyzed by one-way ANOVAs with Tukey's multiple comparison tests. Data represented Mean ± SD. *p<0.05, **p<0.01, ***p<0.001.
Interfering with p62 protein level or function increases cell death induced by mutant huntingtin.Figure 8.(A) GFP-positive cells were scored as live or apoptotic by the appearance of nuclear Draq5 staining. After 48 h of expression, GFP-positive cells attached to the surface with a normal nuclear appearance (open arrowheads) and were scored as live, whereas rounded, partially detached cells with a condensed or fragmented nuclei (arrows) were scored as dead. More than 200 GFP-positive cells in randomly selected microscope views were scored. Bars, 20 μm. (B) The effect of coexpressing either a p62 antisense construct or a deletion construct of p62 lacking the UBA domain on N-HttQ68-induced cell death in HeLa and SHSY-5Y neuroblastoma cells.
Integration of proteomic data into the model.Figure 5Figure 5: Integration of proteomic data into the model. (A) PCA of fluxes from 10,000 random simulations of Yeast7 and 10,000 random simulations of ecYeast7. (B) Flux variability for all non-zero variable fluxes from Yeast7 (3286 reactions, 66.1% of all reactions) and ecYeast7 (3822 reactions, 56.7% of all reactions). (C) Distribution of different pathways in metabolism based on the total usage predicted by ecYeast7 (mg/gDW) and their average flux variability reduction (mmol/gDWh). The discontinuous lines indicate averages of the log values for different metabolic groups. (D) Breakdown of the pathways with the highest average flux variability reduction (mmol/gDWh). Colors correspond to the metabolic groups indicated in (C).
Figure 5(A - C) A549 OTULIN KO cells were transfected with empty vector or different OTULIN constructs as indicated and analyzed by western blot. (D) FLAG-tagged tandem ubiquitin binding entity (TUBE) assay was performed in B cells from control, patient, mother and father to pull down linear ubiquitin linkages. One representative (A - D) of three independent experiments is shown.
Interaction of FGF2 and Spike RBD with immobilised heparin.Figure 1.heparin.Reducing-end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface. PBS running buffer flow rate was 500 µL.min-1. The data for the three sensing channels are reported as an average response. Protein injections are delimited by the red arrows. A Injection of 100 nM FGF2. heparin.Reducing-end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface. PBS running buffer flow rate was 500 µL.min-1. The data for the three sensing channels are reported as an average response. Protein injections are delimited by the red arrows. B 64 nM S1 RBD protein. C 64 nM S1 RBD protein over a streptavidin surface (no heparin functionalization).
Figure 6A. CEM-GFP cells were pre-treated with 50 ng/μL of Vs for 15 min and infected with 0.1 moi of CXCR4-using HIV-1 (NL-4.3) and GFP fluorescence was measured at 488 nm as an indicator of HIV LTR activity. Vs treatment was repeated every 24 h for the experiment. B-D. A similar assay was performed using Jurkat (CD4+ T cell line) and viral replication was assessed by (B) gag RT PCR, (C) p24 ELISA in the culture supernatant, and (D) immunoblotting for p24 (viral capsid protein) in the whole cell lysate. E. U937 (promonocytes) were pre-treated with 50 ng/μL of Vs for 15 min followed by infection with 1 moi of CCR5 using HIV-1 (NL-AD8) and viral replication was measured by gag RT-qPCR at 24 h post-infection (hpi). F. Primary CD4+ T cells purified from human PBMCs (3 healthy donors) were activated, pre-treated with 25 ng/μL Vs for 15 min, and infected with 0.05 moi of HIV-1 NL-4.3. Virus released in supernatant was quantified by p24 ELISA. Vs treatment was repeated every 48 h.
Upon heat shock or proteasome inhibition, DRiPs, polyUb proteins and 20S proteasomes accumulate in PML-NBs (see also -3, EV6 and EV7).Figure 3DRiPs, polyUb proteins (FK1) and PML labelling in HeLa cells treated with OP-puro (25 µM) and heat shock (HS) at 42˚C for 2 h; where indicated cycloheximide was also added (CHX; 50 μg/ml). Scale bars: 5 µm. Lower panel: STED super-resolution microscopy showing colocalization of DRiPs with PML-NBs in PML-GFP HeLa cells treated with a low concentration of OP-puro (5 µM) and heat shock (HS) at 42˚C for 2 h. PML-NBs were visualized using a PML specific antibody, followed by incubation with alexa fluor 647. For convenience the alexa fluor 647 signal is shown in green. Scale bars: 5 µm. PolyUb proteins (FK1) labelling in HeLa cells left untreated or treated with HS at 42˚C for 2 h, alone or combined with CHX (50 μg/ml). Scale bars: 5 µm. STED super-resolution microscopy showing colocalization of endogenous polyUb proteins (FK1) and PML-NBs in PML-GFP HeLa cells treated with HS at 42˚C for 2 h. Scale bars: 5 µm. Quantitation of cells shown in A-C. Number of cells counted/condition: 712 - 1343 in three independent experiments; statistical significance via One-way ANOVA; p < 10-5, ± s.e.m. DRiPs, polyUb proteins (FK1) and PML labelling in HeLa cells treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h; where indicated, cycloheximide was also added (CHX; 50 μg/ml). Asterisks (*) indicate nucleoli. Scale bars: 10 µm. Lower panel: STED super-resolution microscopy showing colocalization of DRiPs with PML-NBs in PML-GFP HeLa cells treated with a low concentration of OP-puro (5 µM) and MG132 (10 µM) for 4 hrs. Scale bars: 5 µm. Quantitation of cells shown in E. Number of cells counted/condition: 298 - 1713 in three independent experiments; statistical significance via One-way ANOVA; p ≤ 0.001, ± s.e.m. Intranuclear distribution of 20S proteasomes in HeLa cells left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Asterisks (*) indicate nucleoli. Scale bars: 10 µm.
Figure 2Validation of HSATIII target introns by qRT-PCR. The graphs show the relative amounts of the intron-retaining (IR) (upper) and spliced (lower) forms in control and HSATIII knockdown cells under three conditions: 37°C (normal), 42°C for 2 h (thermal stress), and thermal stress followed by recovery at 37°C for 1 h. Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the levels in control cells under normal conditions (37°C). Data are shown as the mean±SD (n=3); *p<0.05 (Sidak's multiple comparison test). B Nuclear localization of the intron-retaining RNAs. The relative amounts of intron-retaining RNAs in the nuclear and cytoplasmic fractions were quantified by qRT-PCR and are represented as the ratio (% of the total). GAPDH mRNA and U1 snRNA were used as cytoplasmic and nuclear controls, respectively. Data are shown as the mean±SD (n=3). C Overview of the qRT-PCR analysis of newly synthesized RNA within 1 h after thermal stress removal. CHX, Cycloheximide; EU, 5-Ethynyl uridine D, E The levels of HSATIII target introns in newly synthesized RNAs within 1 h after thermal stress removal, as determined by qRT-PCR. The graphs show the changes in the expression levels of the intron-retaining (IR) (D) and spliced (E) forms. Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the level in the control cells. Data are shown as the mean±SD (n=3); *p<0.05 (multiple t-test modified by Holm-Sidak's method).
– Mutants of cdkb1 and cycb1 show increased number of DSBs and delayed DNA repair upon cisplatin treatment .Figure 2A. Representative examples of comets of 21 days old wild-type plants, cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 double mutant seedlings in full spectrum view of the TriTek Comet Score software. Shown are comets of plants incubated with 50µM cisplatin for one hour and then transferred to medium without cisplatin for 30 min (recovery) and plants incubated without cisplatin for one hour (control), respectively.B. Box plot of percentage of tail DNA of wild-type cells, cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 under cisplatin treatment. Plots are based on analyses of 200 cells per sample from random microscopic fields of three independent biological replications. The percentage of DNA fragments in the comet tail was calculated by the TriTek Comet Score software. The box represents the interquartile range, the line across the box indicates the median values and whiskers represent 5-95 percentile values. Brackets connect plots of sample groups that are significantly different with a confidence level higher than 99.99% calculated with Student's t-test. C. Immunostaining of γ-H2AX foci in wild-type plants and mutant cells after two hours of treatment with 50µM cisplatin.D. Counted number of γ-H2AX foci per cell detected after two hours of treatment with 50µM cisplatin in wild-type and mutant plants. For each sample the γ-H2AX foci of 100 cells were counted and grouped into six categories: cells with no, 1-2, 3-5, 6-10, 11-20 and more than 20 foci per cell.
Figure 3A. Schematic representation of yeast and mammalian β5 subunits and their propeptides. Secondary structure elements, helices (H) and sheets (S), are numbered. The full length human hβ5i (yellow) and β5c (red) as well as the mouse mβ5i (green) subunits cannot substitute the endogenous yeast yβ5 subunit (gray). Expression of the mammalian subunits with their natural propeptide (pp; colored) or with the yβ5 propeptide (pp; gray) is lethal in a yeastpre2Δ knock-out strain (for details see Appendix Supplementary Methods). B. Chimeric β5 subunits are schematically illustrated according to panel A. The N-terminal part of the subunits, which forms the primed and non-primed substrate binding pockets and the active site surroundings, was taken from humanβ5i and the C-terminus from yeast. A construct featuring residues 1-173 from humanβ5i was still lethal. Replacement of the residues 1-139 of yβ5 by the humanβ5i counterpart (aa 1-138; see Appendix Supplementary Note) however was compatible with yeastsurvival. Based on this observation, analogous fragments of the humanβ5c and mouseβ5i subunits were incorporated into the yβ5 subunit to create the respective hβ5c and mβ5i chimeras. C. Subunit β6 significantly contributes to the S3 and S4 pockets of the ChT-L active site. In order to mimic the entire mammalian β5/6 substrate binding channel in yeast we mutated five residues of the yβ6 subunit as well (G89S, K90R, H98Y, T99N and E120Q). These mutations correspond to both the human and the mouse β6 sequence and create two mammalian segments aa 87-101 and aa 108-121 (blue; see also Appendix Supplementary Note). D. The mouse specific point mutation V31M was inserted into the hβ5i/hβ6 chimera to investigate its impact on inhibitor binding. E. Serial dilutions of yeast cells were spotted on YPD plates and grown for 2 days either at 30 °C or 37 °C. Mutant yeasts are significantly retarded in growth and show increased temperature sensitivity. F. Surface illustration of the yCP. The β5 and β6 subunits are depicted in dark gray, with the residue ranges that correspond to mammalian β5 and β6 subunits in the chimeric proteasomes highlighted in gold and blue, respectively. The β5' and β6' subunits of the second β-ring are hidden on the back of 20S proteasome.
Ex vivo stimulation of murine mammary organoids with different PR agonists.Figure 1A Experimental scheme showing the preparation of organoids from mouse mammary glands and ex vivo stimulation with different compounds. B PCA plot of global gene expression profiles from ethanol (CTRL) and R5020-treated murine mammary organoids. C Unsupervised hierarchical clustering of the 500 most variable genes across samples. Normalized expression levels were scaled to Z-scores for each gene. D Volcano plot showing differentially expressed genes (adj. P.Val < 0.05) between ethanol- and R5020-treated mouse mammary organoids, n=3. Genes with log2(FC) > 2 are highlighted in red, genes with log2FC < -2 in blue. Names of genes with -log10 (adj.P.Val) > 10 and abs(log2(FC)) > 2 are indicated. E GSEA showing enrichment of pathways differentially regulated in mouse mammary organoids upon R5020 stimulation, total number of genes = 14,244. The red dashed line indicates NES: Normalized Enrichment Score. F Bar graphs showing Rankl and Wnt4 transcript levels, relative to 36B4 mRNA expression in mammary organoids derived from C57Bl/6 females 6 hours after stimulation with 20 nM R5020 (n=12), DSG (n=6), GSN (n=6), LNG (n=6), CMA (n=6), CPA (n=6), or DSP (n=6). G Bar graphs showing relative Rankl and Wnt4 transcript levels normalized to 36B4 mRNA expression in mammary organoids derived from Balb6/C females 24 hours after stimulation with different progestins, n=3 independent experiments. H, I Bar graphs showing relative Rankl and Wnt4 transcript levels normalized to 36B4 expression in C57Bl6-derived mammary organoids treated for 6 hours with R5020 (H) or LNG (I) and either 10 μM or 100 μM enzalutamide, n=3 independent experiments.
Figure 4(A) PAS staining for total glycogen in skin fibroblasts from different APBD patients. Staining fluorescence was quantified by InCell2200 (see Methods). Means (+/- SEM) are statistically different from each other (p<0.0001, One Way ANOVA). n=3 technical replicates from each patient indicated at the x-axis were used. (B) PAS staining for total glycogen in APBD87 fibroblasts glucose-starved for 48 h (left), or glucose starved and then replenished for the last 24 h to induce glycogen burden (right). Image acquisition performed by Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective and CY3 filter. Scale bar, 100 µm. (C) Image-based multiparametric phenotyping (see Matrials & Methods) of APBD fibroblasts under 48 h glucose-starvation (blue), or starvation and glucose replenishment as in (B) (red). Black line, level of significance p=0.01. Green boxes indicate parameters whose extent of change was increased by glucose supplementation after starvation. (D) Glycolytic (red) and mitochondrial (blue) ATP production determined by Agilent's Seahorse machine and real-time ATP rate assay kit. HC and APBD patient fibroblasts were serum/glucose-starved for 48 h and then full medium was replenished for 24 h without (untreated), or with (chronic) 50 µM 144DG11. Acute, 50 µM 144DG11 was added on assay for 20 min after 24 h of serum/glucose replenishment. Readings were normalized to cell number as determined by Crystal Violet staining. Shown are mean and SEM values based on n=3-6 technical repeats. For all experiments (HC, p<0.01; APBD1, p<0.02; APBD2, p<0.09; APBD3, p<0.11; One Way ANOVA with Sidak's post-hoc correction for multiple comparisons) glycolytic ATP production was increased, as compared to untreated control, by acute, but not chronic, supplementation of 144DG11. Mitochondrial ATP production was increased only by acute supplementation of 144DG11 to APBD2 fibroblasts (p<0.06).