EMBO/sd-character-level-ner huggingface dataset:
Figure 3(A) Growth analysis of strains expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**). Growth curves and calculated doubling times from the exponential phase (insert) are presented. Cells were cultivated in media containing either glucose or glycerol as carbon source. (B) Determination of chronological lifespan via propidium iodide (PI) staining of cells described above. (C) Scheme of the experimental setup to evaluate competitive fitness. (D,E) Analysis of competitive fitness as described in (C). Cor1WT strains harbored a chromosomally-integrated hygromycin selection cassette, whereas Cor1** contained a clonNAT selection cassette (D). Subsequently, selection markers were switched as a control (E).
Negative stain EM results of SARS-CoV-2.Figure 2.(A). Image of negative stained SARS-CoV-2. Nail-like spikes can be clearly seen. Enlarged view of virion boxed in (A). (C) Zoom-in view of a spike boxed in (B). The shape is depicted by red dot line. Length, the diameter of stem and spike's head are 23nm, 4nm and 7nm, respectively. (D). Three-dimensional surface of post-fusion state S2 protein (EMDB code: 9597) [15]. (E). Projection of post-fusion state S2 protein
UBE2SHTH and UBE2R2HTH charged residues contribute to different steps during Ub chain elongation.Figure 3A Schematic of a lysine discharge assay. The acceptor Ub is replaced with free lysine to monitor the stability of the E2~Ub. B Schematic of a *LRLRGG (C-terminus of Ub) peptide transfer to an acceptor UbΔGG, eliminating interactions with the donor Ub. C The E139R substitution decreases the stability of UBE2S~Ub compared to WT, revealed by fluorescent scanning of a lysine discharge assay (top) and its quantification (bottom). Statistical comparison was performed using one-way ANOVA (* p ≤ 0.05, ** p ≤ 0.01, n=4 independent, technical replicates). Error bars: SEM. Data normalized to the initial timepoint of each respective E2~Ub band. D UBE2R2 R149E has a similar E2~Ub stability as WT, monitored by fluorescent scanning (top) of the lysine discharge assay and its quantification through an unpaired Welch's t-test (bottom) (n=3 independent, technical replicates). Error bars: SEM. Data normalized to the initial timepoint of each respective E2~Ub band. E Fluorescent scans (top) and quantification (bottom) of a *LRLRGG peptide transfer assay comparing UBE2S WT and ER/ER reveals no significant difference through an unpaired Welch's t-test (n=3 independent, technical replicates). Error bars: SEM. Data normalized to the maximal *LRLRGG-Ub formation by WT UBE2S. F UBE2R2 R149E is still defective in the transfer of the *LRLRGG peptide to an acceptor Ub compared to WT, probed by fluorescent scanning (top) and its quantitation (bottom). Statistical comparison was performed using an unpaired Welch's t-test (** p ≤ 0.01, n=5 independent, technical replicates). Error bars: SEM. Data normalized to the maximal *LRLRGG-Ub formation by WT UBE2R2.
Figure 3(A) Top, DNA content (n/2n) of exponentially growing (OD600 = 0.6-0.8) smc2-8 yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). Graph: Pkn of YEp13 before and after the inactivation of condensin. Top, DNA content (n/2n) of exponentially growing yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). (B) Experiments conducted as in (A) but in scc1-73 yeast cells. Graph: Pkn of YEp13 before and after the inactivation of cohesin. Top, DNA content (n/2n) of exponentially growing yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). (C) Experiments conducted as in (A) but in smc6-9 yeast cells. Graph: Pkn of YEp13 before and after the inactivation of the Smc5/6 complex. (D, E) Experiments conducted as in (A), but in cells arrested in G1 with alfa-factor (D) or in metaphase with nocodazole (E) for 2 h at 26oC and for one additional hour at 26oC or 35oC. (F, G) Experiments conducted as in (B), but in cells arrested in G1 with alfa-factor for 2 h at 26oC (F) or in metaphase with nocodazole (G) and for one additional hour at 26oC or 35oC. (H, I) Experiments conducted as in (C), but in cells arrested in G1 with alfa-factor (H) or in metaphase with nocodazole (I) for 2 h at 26oC and for one additional hour at 26oC or 35oC.
Comparison of blood gene profiles of infants who experienced post-vaccination fever with those who remained afebrile.Figure 6a, b) Agreement plot. Red = differentially expressed (DE) in febrile infants, blue = DE in afebrile infants, purple =DE in febrile and afebrile infants. c) Exemplar genes differentially expressed at 4 hours and 24 hours, respectively, between infants who experienced a fever within 24 hours of vaccination and afebrile infants. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The false-discovery rate (FDR) was derived by comparing fold-changes in gene expression between infants who experienced post-vaccination fever and those who remained afebrile. P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). Pre-vaccination samples n= 125, 4 hour samples n= 28, 24 hour samples n = 31, 3 day sample n= 30, 7 day samples n= 36. d) Performance of predictive model built with sparse distance weighted discrimination (sdwd) algorithm to predict fever following concomitant 4CMenB vaccine. e) Variable importance score of the features from the sdwd model. f) Expression levels (Limma E value) of the top three transcripts from the sdwd model in the individuals that develop fever (n=33, red circles) and those that don't develop fever (n=21, blue circles); G, H Support Vector Regression (SVR) performance of model to predict post-vaccination MenB-specific SBA titres, g) training dataset (n=36), h) performance of model in the test dataset (n=9) , i) the top 5 genes ranked by importance from the SVR model.
Figure 2(A) Immunofluorescence analysis of the old APPPS1 slice in co-culture using antibody 6E10 to detect amyloid plaques (green) and Thiazine Red (TR, red) to visualize fibrillar amyloid cores. At 7 DIV, the majority of amyloid plaques contain 6E10 and TR positive dense core surrounded by 6E10 positive halo of diffuse Aβ. In contrast, at 14 DIV, the majority of amyloid plaques are core-only plaques co-stained with 6E10 and TR without detectable 6E10 positive halo. Lower panels are higher magnification images of boxed regions. Scale bars: 50 µm. (B) Quantitative analysis of core-only plaques in old APPPS1 slices cultured alone (white bars) or in co-culture (gray bars) at 7 and 14 DIV reveals an increased number of core-only plaques upon co-culturing of old APPPS1 brain slices together with young WT slices. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, each experiment including 3 independent slice culture dishes (***P < 0.001). (C) Western blot analysis of Aβ levels (triplicates) in the old APPPS1 tissue cultured alone or in co-culture with the young WT (14 DIV) using 2D8 antibody.(D) Quantification of Western blot signals reveals decreased levels of Aβ upon co-culturing compared to old tissue cultured alone. The values are normalized to the levels of Aβ in the old tissue cultured alone and represent mean ± SEM from 2 independent experiments, each experiment was performed in triplicates (**P < 0.01).
Figure 3Figure 3.Expression analysis of splicing factor mRNAs in various GBM datasets.A. The genes encoding splicing factors down-regulated by miR-10b are expressed at lower levels in various GBM datasets relative to their expression in normal brain tissues.B. In contrast, many splicing factors up-regulated by miR-10b are overexpressed in the GBM datasets.Six high-content GBMmicroarray datasets from the Oncomine resource (https://www.oncomine.org/resource/main.html), including TCGA_BrainGBM (2), Bredel Brain2 (31), Lee Brain (32), Liang Brain (33), Murat Brain (34) and Sun Brain (35), that collectively contain information for 858 GBM and 52 control samples, have been utilized for the analysis. The data is presented as log2 fold change between GBM and normal brain tissues.
a) Immunofluorescence for LAMP-2A and LAMP-1 of sections from the dorsal motor nuclei of the vagus nerves of brains from two different unaffected control (Ctr) individuals and two individuals with Parkinson's disease with the G/S LRRK2 mutation.figf6(a) Immunofluorescence for LAMP-2A and LAMP-1 of sections from the dorsal motor nuclei of the vagus nerves of brains from two different unaffected control (Ctr) individuals and two individuals with Parkinson's disease with the G/S LRRK2 mutation. Left: images from representative fields. Right: number of fluorescent puncta per cell. Black dots, values in individual cells; red lines, mean. (b) Immunohistochemistry for LAMP-2A in the same samples. (c) Immunofluorescence for LAMP-2A (red) and LRRK2 (green) in the same samples. Merged images from the two fluorophore channels are shown (for colocalization quantification, see Supplementary Fig. 7b). Scale bars, 10 μm. Values are mean + s.e.m.; *P 0.05, ANOVA and Bonferroni test.
Figure 5HeLaS3 whole-cell extract was immunoprecipitated using anti- AGO2 antibody or IgG, as mock IP. TERC RNA enrichment in AGO2 RIP as compared to IgG RIP was assessed by RT-qPCR . 7SK RNA was used for normalization (n=4 experimental replicates). RIP assay was performed from HeLaS3_FH AGO2 and HeLaS3_GFP cell extract using anti-HA antibody or IgG, as negative control, followed by TERC detection by RT-qPCR. 7SK RNA was used for normalization (n=3 experimental replicates). HeLaS3 cells were transduced with a lentiviral vector coding for wild-type TERC (HeLaS3_TERCwt), TERC mutated in position 313-340 (HeLaS3_TERC (313-340mut)) or TERC delated in position 12-31 (HeLaS3_TERC (Δ 12-31)). Whole-cell lysates from HeLaS3_TERCwt, HeLaS3_TERC(313-340mut) and HeLaS3_TERC (Δ 12-31) was immunoprecipitated using an anti-AGO2 antibody in order to assessed the impact of TERC mutations on AGO2 binding. For each experiment the enrichment of ectoTERC as compared to 7SK RNA in RIP samples was normalized on the amount of ectoTERC and 7SK RNA in input samples. Association of AGO2 with TERC(313-340mut and TERC (Δ 12-31) was compared to the association with TERCwt (n=3 experimental replicates).
MCL‐1 interacts with Beclin‐1 at the mitochondria.Figure 4(A) MCL‐1 interacts with Beclin‐1. Lysates from H1299 stably expressing GFP-LC3 were immunoprecipitated using control or MCL‐1‐specific antibodies and analysed for the presence of Beclin‐1 by western blot. (B) H1299 cells stably expressing GFP-LC3 were fractionated into HM (containing mitochondria), and LM (containing ER). Fractions were analysed for the presence of MCL‐1, BCL‐2, Beclin‐1, the mitochondrial marker mtHSP70 and the ER marker calreticulin. (C) MCL‐1 was immunoprecipitated from HM and LM before analysing the presence of MCL‐1 and Beclin‐1.
Figure 2Screening of mitophagy regulators for removing damaged mtDNA. HeLa cells were infected by lentiviral particles containing the indicated knockdown vectors. Five days later, cells were treated with 200 µM H2O2 for 2 h. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima. ShATG5 was used as a negative control. Right panels for each image show the FACS-based mito-Keima dot plots. The y-axis represents the fluorescence emission of mito-Keima at pH 4.0 (lysosome), while the x-axis indicates mito-Keima at pH 7.0 (mitochondria). The percentages of cells within the different regions are indicated. Scale bar, 10 µm. Control or ATAD3B KO HeLa cells stably expressing mito-Keima were treated with 4 mM 3-NPA for 2 h and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (B). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, **p < 0.01. Control or KO ATAD3B HeLa cells were treated with 4 mM 3-NPA for 2 h. Cells were washed with fresh medium and incubated for another 1 h. Cells were then fixed and immunostained with DAPI and anti-8-oxo-dG antibodies and were analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in cells described in (D). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test , N.S., not significant, **p < 0.01.
Fig.Figure 5A Representative overview images and quantification of the β-Galactosidase reporter signal in the DG of 8-week old, 24-week old and 36-week old BATGAL mice. Note the decrease in reporter signal with increasing age (n= 4 animals per age, 200-300 cells per animal) [8 weeks vs 24 weeks p=0.0268, 8 weeks vs 36 weeks p=0.0268]. Scale bar= 100µm. B Representative images of BrdU+ cells (red) expressing DCX (grey) or β-Galactosidase (green) at 14 days post BrdU injection (dpBrdU), 28dpBrdU and 42dpBrdu in 8-week old and 24-week old BATGAL mice. Scale bar= 10µm. C Percentage of BrdU+ cells expressing DCX in 8-week old and 24-week old BATGAL mice (14dpi: n= 3 animals, 28dpi: n= 3 animals, 42dpi: n= 3 animals). Note the prolonged expression of DCX in 24-week old mice [28dpBrdU p=0.0155]. D Percentage of BrdU+ cells expressing the β-Galactosidase reporter in 8-week old and 24-week old BATGAL mice (14dpi: n= 3 animals, 28dpi: n= 3 animals, 42dpi: n= 3 animals). Note the reduced reporter expression in 24-week old mice [28dpBrdU p=0.0117, 42dpBrdU p=0.0034].
Newly synthesized proteins after heat shock.Figure 4A-B Accumulation of newly synthesized proteins after mock shock (A) and heat shock (B). Line graphs showing the accumulation of signal intensity (log2-transformed ratios to pre-shock control) in the soluble fraction. Orange lines show the mean ratio.D Combined violin- and boxplots show the log2-tranformed ratio to pre-shock of newly synthesized proteins in the soluble fraction after heat shock. Proteins that aggregated in the pre-existing (light) fraction are compared to all other proteins. Boxplots indicate median, first and third quartiles with whiskers extended to 1.5 times the interquartile range out from each quartile. Violin plots show the data distribution. Data from at least two biological replicates. Data information: All proteomics data shown in A-D and F is quantified with at least two unique peptides from at least two biological replicates. Proteomics data shown for newly synthesized proteins (heavy). HS = heat shock. MS = mock shock. E Ten proteins with the highest intensity at five hours after heat shock (i.e. strongest upregulation) are listed in order of decreasing intensity. Heat shock proteins are highlighted with red. F Comparison of heat shock-induced protein synthesis and mRNA synthesis after heat shock (Vihervaara et al, 2017). Ten most upregulated proteins in the proteomics analysis are labelled and highlighted in blue; heat shock proteins are labelled red. HSPA1A and HSPA1B could not be distinguished from one another in the proteomics analysis and are plotted as they would have the same intensity (indicated with a vertical dashed line).
Figure 5A Immunoblot analysis of immunoprecipitated Flag-tagged histone H2B using anti-Flag or anti-SUMO antibodies in the indicated mutants, The upper and lower panels show Flag-H2B-SUMO and Flag-H2B-ub, respectively. One, two or three asterisks indicate mono-, di-, and poly-sumoylated histones (upper panel), respectively, and an arrowhead and bullet point represent mono-ubiquitylated H2B, and a non-specific band, respectively. B Immunoblot assay of sumoylated proteins in extracts prepared from the strains Anti-Flag blotting for Flag-H2B was used to verify the similar loading. C Chromatin double immunoprecipitation (ChDIP) of Flag-H2B-ub in the indicated strains expressing Flag-H2B. The first ChIP was performed with anti-Flag agarose, and the eluted samples were immunoprecipitated with an antibody against ubiquitin. PCR signals from the indicated genes were normalized to an internal control and the input DNA. The error bars indicate the SD from three independent chromatin samples. Data information: Asterisks indicate statistically significant differences compared with WT in *, P <0.05; **, P <0.01 D ChDIP of Flag-H2B-SUMO in the indicated strains expressing both Flag-H2B and HA-SUMO. Chromatin samples were sequentially immunoprecipitated with anti-HA agarose followed by anti-Flag agarose. PCR signals were normalized to the input DNA. The error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences compared with WT *, P <0.05; **, P <0.01 E ChDIP of Flag-H2B-SUMO in the indicated mutant strains during CUP1 gene induction The CUP1 induction was performed CUP1 OFF and ON indicate uninduced and induced (5 min) conditions, respectively. The error bars represent the SD from three independent ChDIP assays. Data information: Asterisks indicate significant differences between uninduced and induced cells in (E) (*, P <0.05; **, P <0.01). F Immunoblot assay of Flag-tagged Ulp2 in the indicated strains. Anti-PGK blotting shows similar loading. ChIP assays in the indicated strains expressing Flag-tagged Ulp2 The error bars indicate the SD from three experiments. Data information: Asterisks indicate statistically significant differences compared with WT *, P <0.05; **, P <0.01 ChIP assays in the indicated strains expressing Flag-tagged Ulp2 The error bars indicate the SD from three experiments. Data information: Asterisks indicate statistically significant differences compared with WT *, P <0.05; **, P <0.01 ChIP assays in the indicated strains expressing Flag-tagged Ulp2 The error bars indicate the SD from three experiments. Data information: Asterisks indicate statistically significant differences compared with WT (*, P <0.05; **, P <0.01).
Figure 1(A) Targeting strategy for the conditional disruption of the Clppgene. Map of the genomic locus region of the wild type Clpp gene (wild type), the Targeting Vector, the Clpp locus after homologues recombination (Targeted Allele) and targeted allele after Flp-recombinase-mediated excision of the PuroR cassette (Conditional KO Allele- ClpploxP) and the knocked-out allele after Cre-recombinase-mediated excision of the exons 3-5 (Constitutive KO Allele - Clpp-). The position of primers used for genotyping is indicated by red arrows. (B) Southern Blot analysis of targeted ES cells. Positions of wild type (WT - 14.7kb) and targeted (loxP - 12.8kb) alleles are indicated. (C) Genotyping analysis of wild type (Clpp+/+), heterozygous (Clpp+/-) and homozygous (Clpp-/-) Clpp knockout animals. DNA fragments corresponding to the wild type (WT-199 bp), and knockout (KO - 273 bp) Clpploci are indicated. (D) Analysis of Clpp transcript levels in heart (n=4). Bars represent mean ± standard deviation (SD). Student's t-test was used to determine the level of statistical difference. (E) Western blot analysis of CLPPprotein levels in heart (He), quadriceps (Quad), liver (Li), kidney (Ki), lung (Lu), brain (Br), spleen (Sp) and pancreas (Pa). TUBULIN and ACTIN are used as a loading control. The arrowhead indicates positions of CLPP, and the star indicates position of cross-reacting bands.
Fs are targeted for degradation by APC/CCdh1.Figure 2A Protein levels of E2F7/8 and two known APC/CCdh1 substrates in 293T cells 48 hours after transfection with CDH1-Flag or CDC20-Flag. Immunoblots are representative examples of two independent replicates. B Expression of EGFP-tagged E2F7/8 in 293T cells 48 hours after transfection of flag-tagged CDH1, flag-tagged CDC20 or empty vector. Immunoblots are representative examples of two independent experiments. C Effect of CDH1 depletion on protein levels of E2F7/8 in HeLa cells with stable expression of inducible E2F7/8-EGFP. Overexpression of E2F7 was induced using doxycycline at the onset of release from a thymidine block. D Co-immunoprecipitation of EGFP-tagged E2F7/8 with CDH1-Flag after 48 hours of co-expression in 293T cells. Cells were treated with 10 µM MG132 for 5 hours prior to harvesting to limit immediate proteasomal degradation of E2F7/8 after binding to CDH1. Immunoblots are representative examples of three independent experiments. Asterisks indicate IgG bands; arrow indicates the CDH1-Flag band E HeLa cells with stable inducible E2F7/8-EGFP were imaged by fluorescence and differential contrast (DIC) microscopy at 3 minute intervals. Cells were treated with CDH1 siRNA for 10 hours, synchronized at the G1-S border by 16h thymidine treatment, followed by thymidine release and induction of E2F7/8-EGFP by doxycycline. Mean integrated fluorescence of the cells was measured and normalized to the intensity in the frame of nuclear envelope breakdown (NEBD) (set at 100%), as determined by cytoplasmic dispersal of the fluorescent signal. The x-axis is set to 0 at the onset of anaphase, as observed in the DIC channel. Graphs shown are mean±s.e.m. Left graph: control n=15, Cdh1 RNAi n=14 both from 3 independent experiments. Right graph: control n=13, Cdh1 RNAi n=13 both from 2 independent experiments F Montages of representative cells from experiments shown in (E); time (min) indicates time from onset of anaphase.
a) Knockdown of dRagA and dRagC decreased dS6K phosphorylation (Thr 398).figf1(a) Knockdown of dRagA and dRagC decreased dS6K phosphorylation (Thr 398). Drosophila S2 cells untreated (lane 2) or treated with the indicated RNAi were starved of amino acids for 1 h followed by amino acid stimulation for 30 min Phosphorylation and protein levels of dS6K were determined by immunoblotting with the indicated antibodies. (b) dRagA and dRagC are not required for dAkt phosphorylation. Drosophila S2 cells untreated (lanes 1-3) or treated with the indicated RNAi were starved of amino acids for 1 h and stimulated with amino acids for 30 min. Phosphorylation and protein levels of dAkt were determined by immunoblotting with appropriate antibodies as indicated. NC, negative control RNAi. (c) dRagA and dRagC function in parallel with TSC2 and PTEN. dRagA and/or dRagC RNAi was added to S2 cells in combination with dTSC2 or dPTEN RNAi, as indicated. dTSC2 or dPTEN RNAi treatment increased pdS6K (Thr 398) and this increase was compromised by dRagA or/and dRagC RNAi. Full scans of blots are provided in Supplementary Information, Fig. S6.