EMBO/sd-character-level-ner huggingface dataset:
Figure 3A) Bar graph showing the relative change in OCT-measured volume between the outer nuclear layer and the RPE layer following intervention with test articles. Anti-VEGF-A and anti-ANG-2 were given at 5 mg/kg and anti-VEGF-A/ANG-2 at 1, 5, and 10 mg/kg (low, mid, and high dose).B) Representative OCT images converted to heat maps with the scaling grade given on the right of the figure delineating fluid filled areas of the retina. Baseline (D46) and 14 days treatments of IgG control, anti-VEGF-A, anti-ANG-2, and bispecific anti-VEGF-A/ANG-2 at the end of the experiment (D60) are shown. For the region of interest, the corresponding FA image at D60 is also shown as comparator.
Autophagy is inhibited upon AICD induction through PKA activation.Figure 4A-B. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Wb are representative of at least four independent experiments. Levels of proteins and phosphorylation status of AMPK were quantified and analyzed as shown. In these panels, Time states for "time, hours, after AICD treatment" and indicates at which time upon AICD induction samples have been collected. C. Jurkat cells were transfected with the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of three independent experiments. D. Protein samples from Jurkat cells over expressing empty vector or AMPKS173A-S485A were immunoblotted for LC3 and LC3-II/Actin ratio was quantified and analyzed as shown. Data represent mean ± SE of three independent experiments. E-F. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments. Levels of proteins and phosphorylation status of LC3 were quantified and analyzed as shown. G. Jurkat cells were transfected with the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as described above. H-I. Cells where treated as in (A) and where indicated 10μM H89 was added. Quantitative analysis of P-AMPK Ser485-491 phosphorylation (H) and of LC3-II/LC3-I ratio (I) during AICD are shown. J-K. AICD was induced in Jurkat cells (J) and in hPBT cells (K) in presence of 10μM H89. At indicated time points (hours) after AICD induction, apoptotic cells were detected by flow cytometry as AnnexinV/PI double positive cells and the ratio between AICD and Ctrl values obtained are shown.
NEE-activated cFos-positive neurons are more likely than non-active neurons to bear nuclear infoldings in Satb2 floxed but not in Satb2CamCre mice Representative confocal images (Z-axis projection) of CA1 pyramidal neurons of: (top panel) Satb2 floxed mice (n = 3) maintained in their home cages (HC), (middle panel) Satb2 floxed mice (n = 4) subjected to NEE for 1.Figure 5Representative confocal images (Z-axis projection) of CA1 pyramidal neurons of: (top panel) Satb2 floxed mice (n = 3) maintained in their home cages (HC), (middle panel) Satb2 floxed mice (n = 4) subjected to NEE for 1.5 h, (bottom panel) Satb2CamkCre mice (n = 5) exposed to NE for 1.5 h. Brain sections were stained for cFos and Lamin B2. Scale bars: 10 μm. Higher magnification views of boxed areas in A (middle panel). Arrows show examples of infolded, cFos-positive nuclei of the CA1 field of Satb2 floxed mice subjected to NEE. Scale bars: 5 μm. 2 x 2 contingency tables showing the number of analyzed CA1 pyramidal neuron nuclei grouped by category: cFos+/cFos-, infolded/non-infolded in Satb2 CKO mice (left) and Satb2 floxed mice (right). In floxed animals exposed to novel environment, cFos-positive nuclei were more likely than cFos-negative nuclei to bear infoldings, χ -square test of independence, χ2 (1, N = 3578) = 54.649, p < 0.00001. No significant relationship between cFos expression and nuclear infoldings was identified for the CA1 neurons of Satb2CamkCre mice (χ2 (1, N = 3003) = 0.80, p = 0.3693).
De novo DNA methyltransferases establish neuronal DNA methylation patterns and facilitate neuronal gene expression.Figure 3A, NPCs carrying Dnmt3a/b-KO or WT alleles were differentiated into neurons. Neurons were isolated by FACS and their DNA methylation patterns analyzed using RRBS (n = 3 cultures per genotype). B, We identified 22,150 CpGs that differed in methylation (q < 0.05 and absolute methylation difference > 20 %) between KO and WT neurons, accounting to 4.55 % of all CpGs covered by RRBS being hypomethylated (hypoCpG; violet) and 0.22 % being hypermethylated (hyperCpG; red) in KO neurons. C, Hyper- and hypomethylated CpGs were depleted from CpG islands and promoters, but enriched at gene bodies. Hypomethylated but not hypermethylated CpGs were enriched at enhancer regions (* FDR adjusted p < 0.001, p-values determined by linear regression). D, Hypomethylated CpGs were enriched at neuronal super-enhancers (p < 0.001; hypergeometric test). Depicted are methylation percentages of the 615 hypomethylated CpGs located within neuronal super-enhancers. E, CpGs that gained methylation during neuronal differentiation (Fig. 1), were hypomethylated in KO neurons compared to WT neurons. Depicted are the 441 CpGs covered by RRBS in both datasets. F, Genes containing hypomethylated cytosines (6,275 genes) were significantly enriched in pathways related to neuronal function as annotated in the Reactome Pathway Database. G, Significantly enriched biological processes and cellular components from SYNGO enrichment analysis. H, Dnmt3a/b-dependent loss of DNA methylation at neuronal gene candidates was associated with reduced transcription in neurons. Depicted are mean CpG methylation levels at specified genomic regions as determined by RRBS (left) and expression fold changes in KO versus WT neurons as determined by qRT-PCR (right; n = 4 cultures per genotype; p-values from Mann-Whitney test; depicted are data points for every sample with genotype means ± SEM).
Fig.Figure 5Genes are depicted in their known cellular components, with previously described established GWAS (Efthymiou & Goate, 2017; Verheijen & Sleegers, 2018) in black, and the prioritized novel genes (this paper) in red.
Fig.Figure 2Ensemble of 10 lowest energy NMR-derived structures of apo CaM/IQ complex. Structural statistics are given in Table 3. Energy minimized average structure of apoCaM (cyan) bound to IQ (red), showing an intermolecular salt bridge between IQ K1662 and apoCaM E88. The K1662 side chain amino nitrogen atom is 2.7 Å away from the side chain carbonyl oxygen of E88. Intermolecular hydrophobic contacts between IQ I1654 (red) and apoCaM F90 (cyan). Side chain atoms colored yellow. The I1654 side chain methyl carbon atom is 2.2 Å away from the closest aromatic ring atom of F90. Intermolecular hydrophobic contacts between F1658 of IQ and F90/M110 in apoCaM. The aromatic side chain atoms of F1658 are 2.3Å and 2.6 Å away from the closest side chain atom of F90 and M110, respectively. FP titrations showing binding of apoCaM to IQ peptides WT (black) and K1662E (red) and of apoCaM mutant E88K to IQ WT (green) and IQ peptide K1662E (purple, "charge inversion"; see Table 4 for binding parameters and standard errors). FP titrations showing binding of apo-CaM to IQ peptides WT (black), I1654A (red), and F1658A (green) and of apoCaM mutant F90A to IQ WT (blue; see Table 4 for binding parameters and standard errors).
Figure 5Subcellular membrane fractionation of control and Rab22A-, BLOC-1-, BLOC-2-knockdown HeLa cells and probed the fractions for pallidin, dysbindin and muted. Red colored box emphasizes BLOC-1 membrane association in the respective cell types. Cytosol-membrane fractionation of homogenates from HeLa cells (B as indicated. Protein band intensities were quantified and indicated the percentage membrane association on the gels Cytosol-membrane fractionation of homogenates fro melanocyte as indicated. Protein band intensities were quantified and indicated the percentage membrane association on the gels Pull-down of His-Rab22AWT using HeL lysate the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel Pull-down of His-Rab22AWT usin melanocyte (E) lysate. In E, the beads were preloaded with GTPγS or GDP the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel Pull-down of different His-KIF13A domains using HeLa cell lysate the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel Model illustrating the recruitment (left) and association (middle) of Rab22A, BLOC-1 and BLOC-2 in a sequential manner onto the membrane buds of E/SEs followed by interaction with KIF13A motor. Rab22A-BLOC-1-BLOC-2 complex possibly extend the membrane buds into RE tubules with KIF13A motor (right) movement on microtubules
Figure 4 A, B mRNA expression in scWAT of mice fed a diet with 0.0075% Rosi or control diet for 2.5 weeks, determined by qRT-PCR t-test Cited4-/- vs. Cited4+/+ (Rosi), (A) n=5/5/6/6, **P=0.003 (Ucp1), **P=0.008 (Cidea), **P=0.007 (Cyc1), *P=0.048 (Ndufb3), (B) n=5/4/5/5, *P=0.022 (Ucp1), *P=0.037 (Cidea)  C, D Ucp1 expression in scWAT of mice treated as in A, as determined by Western blot with VCP as loading control t-test Cited4-/- vs. Cited4+/+ (Rosi), females: n=5/5/6/6, *P=0.011, males: n=5/4/5/5  E mRNA expression in scWAT of female mice treated with CL-316,243 (CL) (1 mg/kg/day via Alzet minipumps) or vehicle for 10 days (n=5/7/13, t-test Cited4-/- vs. Cited4+/+ (CL))   F mRNA expression in scWAT of female mice exposed to 5°C or 23°C for 2 weeks (n=5/10/6, t-test Cited4-/- vs. Cited4+/+ (5°C))
a) Wild-type FAK cells were suspended in 1.figf5(a) Wild-type FAK cells were suspended in 1.4% methylcellulose solution in growth media and plated on agarose for 3 days. Top and bottom right, cells were recovered and cytospins prepared that were stained for anti-pTyr-416-Src (red), LC3B (green) and with DAPI (blue) or for paxillin (red), LC3B (green) and with DAPI (blue). Higher-magnification images of the outlined areas are also shown. Solid arrows indicate localization of pTyr-416-Src in LC3B-positive puncta. Scale bars, 20 μm. Bottom left, immunoblotting was carried out on lysates from adherent cells and from cells recovered from methylcellulose using anti-pTyr-397 FAK, anti-FAK, anti-pTyr-416 and anti-Src antibodies. (b) FAK+/+ cells were suspended for 1 h in PBS and cytospins were prepared that were stained for anti-pTyr-416-Src (green) and with DAPI (blue). A higher-magnification image of the area outlined is also shown. Scale bar, 20 μm. (c) LC3B was immunoprecipitated (IP) from adherent or suspended FAK+/+ and then immunoblotted with anti-Src and anti-LC3B antibodies. Lysates were also immunoblotted with anti-pTyr-397-FAK and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.
Figure 1A Human SNCA gene contains six coding exons and two 5' non-coding exons. The exons are represented by vertical colored boxes. The region from exon 1a to exon 2 (~2.5 kb) is scaled up to show the distribution of histone PTMs. Distribution of the histone PTMs from the SN region of one donor brain sample is shown. Peaks of three different histone PTMs, H3K4me3 (green), H3K27ac (blue), and H3K27me3 (black), at the regulatory region of SNCA were adopted from Roadmap Epigenomics Database. For a detailed view, please see Appendix Figure S1 where screenshot of the original figure is shown. The TSS is indicated by a red vertical bar and distances of exon 1a and 1b from the TSS are indicated. B ChIP gel images showing the relative enrichment by H3K4me3 in controls (n=9) and PD patients (n= 18). PCR amplified a 188-bp region of SNCA from intron 1 where H3K4me3 peak was at its optimum. Mouse IgG (mIgG) was used as control and the bands were normalized by unbiased amplification from respective inputs. Data information: Data represent mean ± standard error of the mean. C Relative intensities calculated from the gel images. Graph shows that H3K4me3 was significantly enriched at the upstream regulatory region of SNCA in PD compared to control subjects. ****P < 0.0001. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated for all. Data information: Data represent mean ± standard error of the mean.
Figure 1Time lapse images of T6SS activity in tagged parental strain (VipA-mCherry2) and ∆tssA mutant background. The parental strain forms multiple dynamic structures per cell. ∆tssA mutant predominantly forms dynamic sheath spots (white arrow) and few WT-like sheath structures (blue arrow). Scale bars: 2 µm. H2-T6SS dynamics in tagged parental strain (∆retS TssB2-mCherry2, referred to as WT H2) and ΔtssA2PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. The WT H2 strain harbors multiple dynamic sheath structures per cell, while the ΔtssA2PA strain displays very few dynamic spots or extended and contracting structures. Scale bars: 2 µm.
a) LC3-II levels, which closely correlate with autophagic activity, were higher in mutant lines (expressing Gln74 huntingtin; Q74; lanes 3 and 4) than in wild-type lines (expressing Gln23 huntingtin; Q23; lanes 1 and 2).figf4(a) LC3-II levels, which closely correlate with autophagic activity, were higher in mutant lines (expressing Gln74 huntingtin; Q74; lanes 3 and 4) than in wild-type lines (expressing Gln23 huntingtin; Q23; lanes 1 and 2). Actin was used as a loading control. Data are shown for two independent clonal lines expressing mutant or wild-type huntingtin. Quantification of the band intensity is shown in the right panel. (b) Immunofluorescence analysis of COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin with antibody to LC3 showed more autophagic vacuoles in cells expressing mutant huntingtin that had aggregates (arrow). COS-7 cells without the primary antibody (negative) and COS-7 cells without any treatment (untreated) stained for LC3 are also shown. The numbers of EGFP-positive cells expressing wild-type or mutant huntingtin with (+agg) and without (−agg) aggregates that had higher numbers of autophagic vacuoles (>15-20 vesicles per cell) is shown in the graph. COS-7 cells grown in starvation medium (starvation) or treated with rapamycin (Rap) for 4 h to induce autophagy had more autophagic vacuoles (arrows).
CTDNEP1 and Lipin concentrate at the NE of fly and mouse cells lacking Torsins A - D) Control and dTorKO 5DO fat body cells expressing UAS GFP fusion proteins with the Cg-Gal4 driver, and labeled with anti-Calnexin (red) and a neutral lipid dye (LipidTOX, magenta).Figure 4Control and dTorKO 5DO fat body cells expressing UAS GFP fusion proteins with the Cg-Gal4 driver, and labeled with anti-Calnexin (red) and a neutral lipid dye (LipidTOX, magenta). Note that dTorKO cells are smaller. White arrowhead indicates NE enrichment of LipinmGFP. CTDNEP1Scarlet and anti-Sun1 localization in MEFs. Lipin1mGFP localization in HEK293T cells transfected with a plasmid that co-expresses CTDNEP1myc-WT or -CD (red) and NEP1R1V5 (magenta). (G') Upper: enlargement of panel G, white arrow highlights NE enrichment. Lower: plot showing the intensity of Lipin1mGFP signal (mean ± SD, n = 5 cells) along a 3 µm profile that transects the NE at 0 µm. Mouse Lipin1mGFP expressed in MEFs, red signal shows Calreticulin. Percentage of MEFs with primarily cytosolic or nuclear Lipin1mGFP. Chi-square test. **** p < 0.0001.
Figure 4A,B. To determine the extent to which the p38-mediated innate immunity pathway is activated in nuo-6 and isp-1 mutants, we measured the ratio of phosphorylated PMK-1/p38 to total PMK-1/p38 by Western blotting. Representative blots are shown (A). There is no difference in the ratio of phosphorylated to total PMK-1/p38 in the long-lived mitochondrial mutants compared to wild-type (B). Eight biological replicates of wild-type worms and nine of mitochondrial mutants were quantified. Data information: Error bars indicate SEM. ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison test C. Both long-lived mitochondrial mutants, nuo-6 and isp-1, exhibit markedly decreased food consumption compared to wild-type worms. Six biological replicates per strain were measured, each including at least three technical replicates. Data information: Error bars indicate SEM. ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison test D. Deletion of daf-16 significantly increases food consumption in both wild-type and isp-1 mutants. Three biological replicates per strain were measured, each including at least three technical replicates. Data information: Error bars indicate SEM. ***p<0.001. NS = not significant. Statistical significance was assessed using a two-way ANOVA with Bonferroni posttest E. While disruption of atfs-1 does not affect food intake in wild-type worms, it increases food consumption in nuo-6 worms back to wild-type. atfs-1 loss of function allele was gk3094. Three biological replicates per strain were measured, each including at least three technical replicates. Data information: Error bars indicate SEM. ***p<0.001. NS = not significant. Statistical significance was assessed using a two-way ANOVA with Bonferroni posttest F. Constitutive activation of atfs-1 is sufficient to decrease food consumption to the same level as in long-lived mitochondrial mutants. Three biological replicates per strain were measured, each including at least three technical replicates. atfs-1(lof) allele is gk3094, atfs-1(gof) allele is et17. Data information: Error bars indicate SEM. ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison test
Figure 5A) De novo motifs enriched within 2882 pDHSs determined using HOMER B) Motif distribution in all DHSs ordered by increasing DNase-Seq tag count signal for CD4 TB relative to TN as in Fig 2B. C) DNase-Seq and RUNX1 and ETS1 ChIP-Seq density maps showing the binding at the DHSs as ordered as for (B), with average profiles of RUNX1 and ETS1 binding to the pDHSs in TB compared to TN shown below. D) JUNB ChIP-Seq in TB and TB+ at the DHSs defined in TN and TB and ordered as in (B) with average profiles shown below. E) Patterns of RUNX1, ETS1 and JUNB binding at RUNX, ETS and AP-1 motifs at the Trpm6 locus.
CCN1 regulates NSC pool expansion through EGFR signaling.Figure 6Representative FACS plots showing gating strategy for the analysis of cell types in the adult V-SVZ. Total number of V-SVZ cells isolated from one mouse. n=3, n.s. not significant. Representative FACS plots showing aNSCs (GFP+EGFR+), qNSCs (GFP+EGFR-) and C cells (GFP-EGFR+) in the adult V-SVZ. Percentage of each cell population in the adult V-SVZ. n=3, **p=0.0014 (GFP+EGFR-), ***p=0.0002 (GFP+EGFR+), *p=0.0111 (GFP-EGFR+), ***p=0.0006 (GFP+), n.s. not significant. Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), **p=0.0021 (Ccn1cKO + vehicle vs Ccn1cKO + erlotinib), **p=0.0014 (Ccn1cKO + erlotinib vs Ctrl + vehicle), ****p<0.0001. Densities (units/mm2) of NSC units in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), *p=0.017, **p=0.0061, ***p=0.0002. V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white). Scale bar: 50 μm.
Figure 4A . Expression of geminin-tagged chicken RNase H1-YFP. Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 (X-axis). RNase H1-YFP with or without the geminin degron protein is detected on the Y-axis. The RNase H1-YFP-geminin degron is degraded in G1. In contrast, RNase H1-YFP levels remain stable irrespective of the phase of the cell cycle. 2n and 4n indicate the chromosome number before and after DNA replication B. Bu-1a fluctuation analysis of two independently derived primpol BU-1A(GAA) 10: Gg RNase H1-YFP-geminin degron clones. Since the expression of the RNase H1-YFP-geminin degron construct is not stable (unlike the RNase H1-YFP construct without the degron), Bu-1a expression was assessed separately in the YFP +ve and YFP -ve cells within each clone. Statistical differences calculated the Kruskal-Wallis test. For all panels, at least 36 individual clones were analysed; mean ± SD reported. **** p ≤ 0.0001, *** p ≤ 0.001, ns = not significant C. DRIP-qPCR for R-loops around the engineered +3.5 (GAA) 10 repeat in BU-1 in different phases of the cell cycle. The location of the qPCR amplicons is indicated in the map at the top of the panel. The BU-1 DRIP signal was normalised to -0.5 kb amplicon in G1-arrested cells (t = 0 h) Black: wild type; red: primpol. Error bars = SD. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 D. Workflow for the S9.6-independent detection of newly synthesised R-loops E. Validation of analysis of nascent DNA:RNA hybrid formation in BU-1 locus. Enrichment of 4SU labelled RNA moiety of DNA:RNA hybrids was calculated relative to input in three independent asynchronous wild type (black) or primpol (red) cells, with or without exogenous RNase H treatment. Error bars = SD. ** p ≤ 0.01, * p ≤ 0.05, ns = not significant; unpaired t-test F. Synchronisation and 4SU pulse labelling scheme to identify nascently formed DNA:RNA hybrids G. Newly synthesised R-loops in BU-1 during S phase in wild type (black) and primpol (red). Error bars represent 1SD of three biological repeats of the experiment. *** p ≤ 0.001, * p ≤ 0.05; unpaired t-test
The catalytic activity is essential for Jmjd2a/c functions.Figure 7A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (A) WB. A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (B) RT-qPCR analyses A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (C) serial plating for the generation of growth curves.
PI5P4K2s Localize on Autophagosomes and Regulate Autophagy (A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells transfected for 5 days with two rounds of control, PI5P4K2A, 2B, or 2C siRNA either left untreated or treated with BAF (200 nM, 16 hr) (mean ± SEM).Figure 3(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells transfected for 5 days with two rounds of control, PI5P4K2A, 2B, or 2C siRNA either left untreated or treated with BAF (200 nM, 16 hr) (mean ± SEM). (C) HeLa cells stably expressing GFP-mRFP-LC3 treated as in (A) were analyzed on a Cellomics ArrayScan system as previously described (mean ± SEM). (D) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA and transfected with HA-ATG12 and ATG5 for the last 16 hr (mean ± SEM). (E and F) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, or catalytic-dead PI5P4K2C along with RFP-LC3 for 16 hr (E) or 30 hr (F) were fixed and imaged on a confocal microscope. Bar, 10 μm.(G) Quantification of cells (percentage of total) showing more than ten autophagic vesicles (RFP-LC3 vesicles) in the different conditions from (F) is shown in the graph; n = 200 cells (mean ± SEM). See also Figure S3.