EMBO/sd-character-level-ner huggingface dataset:
Figure 2(A) Confocal imaging showed anti-GA antibody treatment reduces poly-GA aggregates and TDP-43 mislocalization in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP-43 in GFP-positive and -negative cells, respectively. Scale bar denotes 30 µm. (B) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced (donor), non-transduced (receiver) neurons. Four groups were excluded due to very high GFP transduction rate (GFP-negative donor) and very low GFP transmission rate (GFP-positive receiver with IgG and anti-GA) and complete prevention of GA-RFP transmission of anti-GA immunodepletion (GA-GFP receiver with anti-GA). n=3 biological replicates. In total 280 donor GFP, 284 receiver GFP with IgG, 317 receiver GFP with anti-GA, 277 donor GA175-GFP, 294 receiver GA175-GFP with IgG, 311 receiver GA175-GFP with anti-GA cells were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001. (C) Immunoblotting of poly-GA immunoprecipitated from conditioned media using antibody-coupled beads and antibody leftover pre and post immunoprecipitation in the conditioned media. (D) Poly-GA levels in conditioned media before and after immunodepletion with control IgG and anti-GA antibody were determined by immunoassay. n=3 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. ** denotes p<0.01, and *** denotes p<0.001.
Figure 4(A and B) Peritoneal macrophages from Atx+/+ (A) and Atx∆ΜΕ/∆ΜΕ (B) mice were stimulated with LPS (20 ng/mL, 20 min), followed by fixation in 4% paraformaldehyde for 15 min at room temperature and staining with TLR4-anti rabbit FITC (Green), early endosomal marker EEA1-anti mouse Dylight405 (Blue), and Alexa Fluor 594-CTXB (Red). TLR4 internalization was examined with confocal microscopy. Presented are the representative from 3 independent experiments in which more than 95% of the cells exhibited similar results. Scale bar, 10 μm.
Figure 1(A) Schema of the classic HPA axis. CRH causes the secretion of ACTH and cortisol. (B) In the CRH test, the secretion of these hormones is measured after CRH administration. (C) Patients suffering from major depressive disorder (MDD) show a blunted ACTH response to CRH (black line, N=10), compared with control (gray line, N=10) - data from (von Bardeleben et al, 1988), shown are mean ± SEM. (D) Patients suffering from anorexia and admitted to treatment show a blunted ACTH response and hypercortisolemia, which resolves within 6-24 months after weight normalization- data from (Gold et al, 1986a). However, 3-4 weeks after weight normalization, cortisol dynamics are normal whereas ACTH dynamics are blunted. Pregnancy is associated with elevated cortisol levels due to CRH secretion by the placenta. 3 weeks after delivery, cortisol levels and dynamics return to normal, whereas ACTH dynamics are blunted- data from (Magiakou et al, 1996). After 12 weeks, ACTH dynamics normalize as well, while cortisol dynamics remain normal. Individuals recovering from alcohol abuse show hypercortisolemia and blunted ACTH response after admission - data from (von Bardeleben et al, 1989). After 2-6 weeks, these individuals show normal cortisol dynamics, but blunted ACTH responses persist. In all panels, control patient data are denoted by thin gray line (Anorexia: N=13. Pregnancy: N was unspecified. Alcohol abuse disorder: N=11), and case data by a thicker black line (Anorexia: left panel, N=9, center panel, N=5, right panel, N=6. Pregnancy: N=17. Alcohol abuse disorder: N=20). Shown are mean ± SEM for all panels.
a) Akt/GSK-3β/mTOR/P70S6K phosphorylation assessed by western blots after thioperamide administration and OGD/R.f4(a) Akt/GSK-3β/mTOR/P70S6K phosphorylation assessed by western blots after thioperamide administration and OGD/R. LiCl (5 mmol l−1 immediately at reperfusion) was used to rephosphorylate GSK-3β/mTOR/P70S6K. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00. (b) Representative western blots showing the phosphorylation of Akt/GSK-3β/mTOR/P70S6K signaling in H3R−/− mice after OGD/R injury. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00. (c) Representative images showing LC3-positive autophagic vacuoles (red) with 4',6-diamidino-2-phenylindole staining (blue) after administration of thioperamide and LiCl with OGD/R (scale bar, 10 μm). (d) Cell viability assessed by MTT when thioperamide and LiCl were administered after OGD/R (n=7, **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test). (e) Representative western blots and bar graph showing the expression of LC3-I and LC3-II in steady-state autophagy and autophagic flux under OGD/R injury when thioperamide (THIO) and immepip (IMME) were administered (n=6; **P0.01, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 11.
Figure 3(A) Quantification of the fraction of cells with ANKRD26 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m. (B-D) Representative images of WT or ANKRD26-/- PLK4Dox cells immunostained with the indicated antibodies. Scale bar = 5 µm. (E) Quantification of the fraction of WT or ANKRD26-/- hTERT RPE1 cells with cilia. Each dot displays measurements from a single experiment. Data acquired across n = 5 biological replicates. Mean ± s.e.m. (F) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m. (G) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m. (H) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. (I) Quantification of pro-CASP2 levels following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. Each dot displays measurements from a single experiment. Data acquired across n = 6 biological replicates. Mean ± s.e.m.
Figure 6(A) Example of Western blot analysis of DCLK1 and DCX levels in protein extracts obtained from DIV1, DIV7, DIV14 and DIV21 hippocampal neurons. (B) Representative image of a hippocampal neuron (DIV14) immunostained for DCLK1 and β-III-tubulin. Scale bar = 20 µm. (C, D) Representative image of a COS-7 cell transfected with DCLK1-GFP (C) or DCLK1(ΔKD)-GFP (D) and stained for detyrosinated, tyrosinated or α-tubulin.(E) Representative image of a COS-7 cell transfected with DCLK1-GFP and TagRFP-EB3 and recorded during live-cell imaging. Representative GFP was drawn (over a region marked with white boxes) showing that DCLK1-GFP colocalizes with EB3-TagRFP-T. Scale bars = 20 um.
Figure 1(A) Chromosome 7 of S. bayanus is incompatible with the S. cerevisiae genome. The chromosome 7 replacement stain (Sc+Sb-chr7) exhibited serious growth defects when grown on the non-fermentable carbon source (glycerol). The respiration defects could be partially rescued when a whole set of S. bayanus chromosomes were provided (Sc+Sb-chr7 x Sb-ρ⁰), or fully rescued with the addition of S. bayanus mtDNA (Sc+Sb-chr7-ρ⁰ x Sb). Cell cultures were serially-diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed. Sc, S. cerevisiae. Sb, S. bayanus. ρ⁰, mtDNA-less strains. (B) Ectopically-expressing Sc-CCM1 rescues the respiration defect of Sc+Sb-chr7. Spot assays for the wild-type S. cerevisiae (Sc) and the chromosome 7 replacement stain (Sc+Sb-chr7) carrying an empty plasmid (+ vector) or the plasmid with the S. cerevisiae CCM1 gene (+ Sc-CCM1). (C) Sb-CCM1 incompatibility could only be rescued in the presence of S. bayanus mtDNA. The native CCM1 ORF in S. cerevisiae was replaced by either Sb-CCM1 (Sc+Sb-CCM1) or Sc-CCM1 (Sc+Sc-CCM1, as a control) coding regions. Crossing the Sc+Sb-CCM1 strain with the mtDNA-less Sb-ρ⁰ strain (Sc+Sb-CCM1 x Sb-ρ⁰) could not rescue the growth defect. (D) CCM1 incompatibility is symmetric between S. cerevisiae and S. bayanus. The CCM1 ORF in S. bayanus was replaced by either Sc-CCM1 (Sb+Sc-CCM1) or Sb-CCM1 (Sb+Sb-CCM1, as a control) coding regions. Only the Sb+Sc-CCM1 strain exhibited respiration defects and the defect could not be rescued even when a whole set of S. cerevisiae chromosomes were provided (Sb+Sc-CCM1 x Sc-ρ⁰). (E) CCM1 incompatibility probably occurred during the divergence between S. bayanus and the common ancestor of S. cerevisiae, S. paradoxus, S. mikatae and S. kudriavzevii. The endogenous CCM1 in S. cerevisiae was replaced with its orthologous alleles from other Saccharomyces sensu stricto yeasts. Only the strain carrying S. bayanus CCM1 displayed growth defects on glycerol-containing plates.
Figure 2We utilized exome sequencing data from 24 MSI CRCs and corresponding normals and whole genome sequencing data from 12 MSI CRCs and corresponding normals. The tumor data was filtered against the corresponding normals as well as outside controls to remove germline variants. Only the areas targeted by the exome sequencing kit were included in the analysis. Insertions and deletions were then filtered out, leaving the somatic missense, nonsense, synonymous and non-coding mutations in the region targeted by the exome sequencing kit. On this set of variants a) hot spot analysis was performed, and b) MutSigCV was utilized. In the hot spot analysis, genes with non-synonymous or splice site hot spot changes - mutations residing in either the same or two adjacent codons, or two bases flanking an exon-intron boundary - were identified. Previously reported hot spot genes were omitted. The resulting list consisted of 90 somatic hot spots in 88 genes. The MutSigCV analysis included six previously reported hot spot genes, which were then added to the hot spot list. The hot spot list entering validation therefore consisted of 97 somatic hot spots in 94 genes, and these 97 hot spots were re-sequenced with MiSeq sequencing in the validation set of 93 additional MSI CRCs. The data was filtered against > 60,000 outside controls to remove germline variants. Insertions and deletions were left out, resulting in a set of somatic missense, nonsense, and synonymous changes with MAF < 5 x 10-5. In the MiSeq data, 11 of the 94 genes contained additional hot spot mutations
Figure 1Relative expression of miR-183 and miR-96 in GAS, TA and SOL muscles of WT and DKO mice (n=5). SDH staining of GAS and SOL muscles in WT and DKO mice (n=5). Scale bars: 100 μm. Representative photographs of GAS and SOL muscles dissected from WT and DKO mice (n=5). Scale bar: 1cm. ATPase staining of GAS and SOL muscles of WT and DKO mice (left). Corresponding percentage of type I fibers were determined (right) (n=5). Scale bars: 100 μm. Relative mRNA expression of MHC isoforms in GAS muscles of WT and DKO mice (n=3). Oxygen consumption rate of WT and DKO mice in light and dark phases (n=5). Relative mRNA expression of PGC1α and PLIN5 in GAS muscles of WT and DKO mice (n=3 for PGC1α analysis; n=5 for PLIN5 analysis). Relative mRNA expression of PGC1α and PLIN5 in C2C12 cells transfected with miR-183 and miR-96 agomirs (Ago-183/96) or antagomirs (Ant-183/96) (n=3). Western blot analysis of PLIN5 in C2C12 cells transfected with Ago-183/96 or Ant-183/96. Mitochondrial respiration profile of C2C12 cells transfected with Ago-183/96 (J) or Ant-183/96 (K). Cellular oxygen consumption rate (OCR) was measured in real time and corresponding basal and maximal respiration were analyzed by overall OCR (n=3 or 4 per group).
BI‐1 deficiency enhances autophagy flux.Figure 2(A) BI‐1 WT and KO MEFs were treated with EBSS (left panel) or glucose/serum‐free RPMI media (right panel) for the indicated time points. Then, levels of LC3 were determined by western blot analysis. LC3‐I and LC3‐II forms are indicated. Hsp90 levels were assessed as loading control. (B) Quantification of LC3‐II levels relative to Hsp90 expression was performed in several experiments performed as presented in (A). (C) Cells were pre‐treated with a cocktail of lysosomal inhibitors (200 nM bafilomycin A1, 10 μg/ml pepstatin, and E64d; left panel) or 10 mM 3‐methyladenine (3‐MA; right panel) for 12 h and then exposed to starvation. LC3 levels monitored by western blot (D) and quantification of LC3‐II levels relative to Hsp90 were performed in the experimental conditions described in (C). (E) Basal autophagy flux was monitored in cells treated with a cocktail of lysosomal inhibitors (Lys. Inh.) in the presence of normal cell culture media. Right panel: quantification of independent experiments is presented. (F) BI‐1 KO cells were stably transduced with retroviruses expressing BI‐1-GFP or empty vector, and then levels of LC3‐II were assessed over time by western blot after exposure to EBSS. Right panel: as control, the levels of BI‐1-GFP were monitored by western blot. Hsp90 levels were used as loading control. In (B, D and E) mean and standard deviation are presented. Two‐way ANOVA was applied to analyse statistical significance. In parenthesis, the number of independent experiments for each time point is indicated. Student's t‐test was also used in (E) to analyse the statistical significance between each time point (*P0.001). In (B, D and E), normalization was performed as a ratio with the LC3‐II/Hsp90 normalized levels from non‐treated BI‐1 WT cells.
Figure 4(A) Venn diagrams showing up- and down-regulated genes and TEs in the epcr1/2 and arid2/3/4 mutants relative to the wild type, as determined by RNA-seq. The data are from three independent biological replicates. All the overlaps are highly significant (P<2.610-30) as determined by hypergeometric test. (B) Heat maps showing differentially transcribed genes and TEs in the arid2/3/4 and epcr1/2 mutants relative to the wild type. (C) Distribution of up-regulated TEs in nrpe1, epcr1/2, and arid2/3/4 on the Arabidopsis chromosomes. (D) Genome browser snapshots showing the transcript patterns of ROS1 and IBM1 in arid2/3/4 and epcr1/2 relative to the wild type. (E) Effects of arid2/3/4 epcr1/2 on the transcript levels of ROS1 and IBM1-L as determined by qPCR. The expression of ROS1 and the longer version of IBM1 (IBM1-L) was evaluated by qPCR. ACT7 was amplified as an internal control. Bars represent SD from two independent experiments, each with three technical replications.
Model for yNap1 as a H2A-H2B transport and chromatin assembly factor.Figure 7Figure 7 - Model for yNap1 as a H2A-H2B transport and chromatin assembly factor. H2A-H2B synthesis in the cytoplasm results in binding to yNap1. The yNap1-H2A-H2B complex oligomerizes such that the NLS becomes accessible to the karyopherin Kap114p, resulting in nuclear transport. Presumably the oligomer needs to be disassembled for nuclear transport, a function that could be provided by phosphorylation (eg. CK2). In the nucleus, yNap1 deposits H2A-H2B onto hexasome intermediates. After release of H2A-H2B, apo yNap1 can oligomerize through a region involving the NLS. Burial of the NLS results in cytoplasmic localization.
Accelerated molecular dynamic simulation of the rhodopsin dark state: WT, G90D2.Figure 3Accelerated molecular dynamic simulation of the rhodopsin dark state: WT, G90D2.57, T94I2.61 and T94I2.61 with protonated E1133.28. In the absence of the G90D2.57 and T94I2.61 dark state structure, both aspartate and isoleucine were placed in the WT (1GZM [48]) using a favorable rotamer without introducing major clashes with the rest of the protein. However, both isoleucine and aspartate in the dark state structures introduced clashes with several amino acids and were therefore optimized using energy minimization before simulation. WT simulation was done with the protonated SB while in the G90D2.57 system, both E1133.28 and SB were protonated [36]. The protonated state of E1133.28 was unknown in T94I2.61, therefore simulation were done for both deprotonated and protonated E1133.28. The T94I2.61 dark state conformation with deprotonated E1133.28 was similar to that of the WT and was stable, whereas the dark state of G90D2.57 and T94I2.61 with protonated E1133.28 was unstable with a concomitant movement of E1133.28 residue away from the SB, thereby, causing movement of TM3 and opening of the E1133.28-SB activation switch.In contrast to the crystal structures the orientation of the retinal -ionone ring in the simulations was variable with respect to the polyene chain. This indicates a degree of positional freedom within the hydrophobic binding pocket.
Figure 3A. Schematic of the experiments shown in (B), (C), (D) and (E). Bar plot showing the mean of (B) centromere length (n = 4) as determined by whole genome sequencing. Error bars represent the SEM of four independent experiments. Acrocentric chromosomes 13, 14, 21 and 22 were marked by a line as we could not assign the respective reads. Dashed lines indicate the mean. Bars were labeled with asterisks according to the significance of their difference from the mean (t-test). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Bars represent the sum of the length Bar plot showing the mean of (C) CENP-B boxes counts (n = 4) as determined by whole genome sequencing. Error bars represent the SEM of four independent experiments. Acrocentric chromosomes 13, 14, 21 and 22 were marked by a line as we could not assign the respective reads. Dashed lines indicate the mean. Bars were labeled with asterisks according to the significance of their difference from the mean (t-test). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Bars represent the sum of counts of different HOR arrays CENP-B boxes counts are normalized to the average number of mapped reads in each replicate. D. Schematic using representative images of the mFISH labeling followed by CENP-B box FISH method used to identify and quantify centromere specific CENP-B boxes signal in (E). Scale bar represents 10 µm. E. Box and whisker plots of normalized CENP-B boxes intensity over the mean on metaphase spread from three independent experiments (n>50 cells) using the Tukey plot. One-way ANOVA with post-hoc Tukey's multiple comparison test shows high diversity between chromosomes. t-test against the mean was used to estimate the statistical significance for each chromosome. **p < 0.01; ***p < 0.001; ****p < 0.0001.. Scatter plot showing a non-significant or significant negative correlation between the mean of (F) centromere length (n = 4) and mis-segregation rate (from Fig. 1F) (r = Spearman rank coefficient). Lines represent the linear regression with a 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis. Scatter plot showing a non-significant or significant negative correlation between the mean of (G) centromere CENP-B boxes FISH signal (n>50 cells) and mis-segregation rate, respectively (r = Spearman rank coefficient). Lines represent the linear regression with a 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis.
linked polyubiquitination transition on MAVS Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or Lys to Arg mutants together with Myc-Ub and HA-RNF34.Figure 4Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or Lys to Arg mutants together with Myc-Ub and HA-RNF34. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results. Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or 4KR mutant together with Myc-Ub and HA-RNF34. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Ub-K27 antibody. Levels of the transfected proteins were analyzed by performing immunoblotting with an anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results. (C) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or K311R mutant together with HA-RNF34 and/or Myc-TRIM31 following a 3MA (0.2 mM) treatment. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Flag, anti-Ub-K63 or anti-Ub-K27 antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA or anti-Flag antibody. ell-based studies were performed independently at least three times with comparable results. (D) Co-immunoprecipitation analysis of the ubiquitination of endogenous MAVS in THP-1 cells transfected with siCtrl, siRNF34, or siTRIM31 oligos and infected with VSV for the indicated times. Cell-based studies were performed independently at least three times with comparable results. (E) Co-immunoprecipitation analysis of the endogenous interaction of MAVS-TRIM31 and MAVS-RNF34 in PBMCs infected with VSV for the indicated times. Cell-based studies were performed independently at least three times with comparable results.
The loss of ATG13 and ATG101 result in an accumulation of ATG9A at large clusters of p62/SQSTM1.Figure 4(A) Endogenous p62/SQSTM1 level in HCT-116 ATG9A-HA KI-ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO clones was measured by immunoblotting with indicated antibodies. Cells were grown in full DMEM media, treated with or without 100 nM Bafilomycin for 24 hrs and whole cell lysates were subjected to immunoblotting (left). The graph on right shows quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right). (B) Endogenous p62/SQSTM1 level in HEK-293T ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO clones was measured by immunoblotting with indicated proteins. Cells were grown in full DMEM media, treated with or without 100 nM Bafilomycin for 24 hrs and whole cell lysates were subjected to immunoblotting. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right). (C) Confocal images of ATG9A colocalization with p62/SQSTM1. HCT-116 ATG9-HA KI-ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO cells were grown in full DMEM media, fixed, labeled with antibodies for HA and p62/SQSTM1 and imaged (Scale bar 10 µm). ATG101 KO was stained and quantified separately. Images are from the same single independent experiment (D) Quantification of average surface area of p62/SQSTM1 puncta in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates. ATG101 KO was stained and quantified separately. Mean ± SEM, n=1 independent experiment with 30 technical replicates. A break was inserted in the Y axis to accommodate the wide range of p62/SQSTM1 puncta sizes. (E) Quantification of ATG9A colocalization with p62/SQSTM1. Mean ± SEM, n=1 independent experiment with 30 technical replicates. ATG101 KO was stained and quantified separately. Mean ± SEM, n=1 independent experiment with 30 technical replicates.
Figure 3(A) Comparison of pA-DamID and DamID data for for various lamins across a representative genomic locus in HAP-1 and hTERT-RPE cells. Arrow indicates a region with dissimilar pA-DamID and DamID scores. (B) Genome-wide correlation of Lamin B1 pA-DamID and Lamin B1 DamID data. The blue line represents a linear model. The red dashed line represents the diagonal. (C) Venn Diagram showing the overlap in LADs defined by a Hidden Markov Model. (D) Multidimensional scaling (MDS) plot showing all individual replicates of DamID and pA-DamID experiments for various lamins and cell types.