EMBO/sd-character-level-ner huggingface dataset:
Figure 4A Schematic diagram shows how the SunTag-JARID1A system is recruited at the SNCA promoter. The dCas9-5xGCN4, scFV-JARID1A, and sgRNA plasmids are co-overexpressed in the cells. The dCas9-5xGCN4 is recruited to the SNCA promoter as directed by the specific sgRNA. Five scFV-JARID1A molecules in turn recognize the GCN4 polypeptide sequences of dCas9. Upon recruitment of the entire system, SunTag-JARID1A demethylates H3K4me3 at the target region. B Structure of pLvx-scFV-sfGFP-JARID1A. The scFV-sfGFP-catalytic domain of JARID1A was sub-cloned into a lentiviral vector. The distance between the 5' and 3' Long Terminal Repeats in the vector is 8.5 kb. The components of the plasmids are as follows: , RRE, rev response element; cPPT, central polypurine tract; pCMV IE, immediate early cytomegalovirus promoter; scFV, single chain variable fragment; sfGFP, super folder GFP; JARID1A; catalytic domain of JARID1A; GB1, solubility tag protein; NLS, nuclear localization signal; WPRE, woodchuck hepatitis virus (WHP) posttranscriptional regulatory element. C Relative positions of the nine sgRNAs. A ray diagram exhibits the locations of the nine sgRNAs in relation to the upstream regulatory regions of SNCA and the H3K4me3 and H3K27me3 peaks. The locations of the sgRNAs are as follows with respect to the TSS: sgA, 1,117 bp; sgB, 836 bp; sgC, 747 bp; sgD, 700 bp; sgE, 153 bp; sgF, 1,454 bp; sgG, 1,537 bp; sgH, 132 bp; sgI, 1,320 bp. The two non-coding exons (1A and 1B) along with the first coding exon (exon 2) are also shown. GRCh37/hg19 contig was used to describe the location of the gene and associated distribution of histone PTMs. The histone peaks shown were from midbrain regions of two adult postmortem human samples.
Figure 6A. HeLa cells were transfected with the indicated si-RNAs for 24 h and then exposed to hypoxia for 24 h or not. Cells were fixed and immunostained by anti-TOM20 and anti-LC3 antibodies. The colocalization of LC3 and mitochondria was quantified and listed on the right side. Bar = 5 µm. B and C. HeLa cells were transfected with the indicated si-RNAs for 24 h and then exposed to hypoxia for 24 h. Cells were then treated with or without 50 nM BAF A1 (Bafilomycin A1) for 6 h before harvesting. Cell lysates were immunoblotted with the indicated antibodies. D and E. Quantification of the TIM23:Actin and TOM20:Actin ratios from (B and C). All data are from 3 independent experiments. F. HeLa cells were transfected by different truncated FUNDC1-MYC. About 24h post-transfection, cell lysates were immunoblotted using indicated anti-bodies.
Deletion of de novo DNA methyltransferases does not abolish the pro-neurogenic effect of environmental enrichment but modifies hippocampal excitability.Figure 6A-B, Quantification of adult-born (BrdU-positive) cells in the dentage gyrus (DG) of Dnmt3a/b-KO and WT mice after five weeks or three months of environmental enrichment (ENR) or standard housing (STD). BrdU was injected 4.5 weeks before analysis. Depicted p-values correspond to genotype effects from two-way ANOVA. Influences of ENR were significant with p < 0.01. Depicted are data points for every animal with group means ± SEM. C, Bright-field images of BrdU-positive cells from WT and KO mice housed in ENR for three months. Scale bar: 100 µm. D, Fluorescence images of c-Fos-positive cells in the DG and CA3 of WT and KO mice. Scale bar: 200 µm. E, Numbers of c-Fos-positive cells in the DG without (STD) and with acute stimulation in ENR for 1 h (n = 2, WT STD; n = 2, KO STD; n = 5, WT ENR; n = 5, KO ENR). F, Numbers of c-Fos-positive cells in suprapyramidal and infrapyramidal blades of the DG after acute stimulation in ENR (n = 5 mice, WT; n = 5 mice, KO). G, Percentage of new-born neurons (BrdU/NeuN-positive) that expressed c-Fos after ENR (n = 5 mice, WT; n = 5 mice, KO). H, Representative fluorescent image of c-Fos expression in BrdU/NeuN-positive cells in the DG. Scale bar: 25 µm. I, Percentage of c-Fos-positive cells that expressed Dcx in WT and KO mice (n = 5 mice, WT; n = 4 mice, KO). J, Representative fluorescent image of c-Fos-positive cells and Dcx-positive cells in the DG. Scale bar: 100 µm. K, Reduced numbers of c-Fos-positive cells in CA3 of KO mice (n = 4 mice, WT; n = 5 mice, KO). L, Fluorescent image of c-Fos expression in Parvalbumin (Pv)-positive interneurons in CA3. Scale bar: 50 µm. M, A higher percentage of Pv-positive interneurons in CA3 expressed c-Fos in KO mice (n = 4 mice, WT; n = 4 mice, KO).
Fig.Figure 5A. Schematic outline of the experimental design. B. Scheme for WGCNA analysis. C. Upper panel: Expression of RNA module 1 among the three experimental groups. Kruskal-Wallis test. Lower panel: Gene ontology analysis of genes that are part of RNA module 1. In the boxplots in C, the center line indicates the median, while the upper and lower lines represent the 75th and 25th percentiles, respectively. The whiskers represent the smallest and largest values in the 1.5× interquartile range. D. Network representing top 30 hub genes of the gene network based on RNA module 1. E. H3K4me3 at TSS region of genes from RNA module1. Inset shows statistical comparisons among groups (One-way ANOVA, ***P<0.001, ****P<0.0001). Bars and error bars indicate mean ± SEM. Number of replicates analyzed for H3K4me3 profile at TSS (Wildtype vehicle: 3; CamkIIδc TG-vehicle n = 4; CamkIIδc TG-Vorinostat n = 4). F. Upper panel: RNA module 2 and its expression among the three experimental groups. Kruskal-Wallis test. Lower panel: Functional annotations of the genes that are part of RNA module 2. Wildtype vehicle: n = 10; CamkIIδc TG-vehicle n = 9; CamkIIδc TG-Vorinostat n = 9. In the boxplots in F, the center line indicates the median, while the upper and lower lines represent the 75th and 25th percentiles respectively. The whiskers represent the smallest and largest values in the 1.5× interquartile range. G. Gene correlation network of the top hub genes (n =30) of RNA module 2. H. Left panel: Schematic outline of the analysis of microRNA-sequencing data. Right panel: Expression of microRNA module 2 amongst experimental groups. Kruskal-Wallis test. Wildtype vehicle: n = 9; CamkIIδc TG-vehicle n = 7; CamkIIδc TG-Vorinostat n = 10; In the boxplots in H, the center line indicates the median, while the upper and lower lines represent the 75th and 25th percentiles, respectively. The whiskers represent the smallest and largest values in the 1.5× interquartile range. I. Heatmap showing significant negative correlation (FDR < 0.05) between microRNA members of microRNA module 2 and hub genes from RNA module 2 (see panel G).
AID expression in pancreas promotes DNA lesions.Figure 3A, B Analysis of AID mutagenic activity in Elastase1 (A) and Elastase2a (B) by next-generation sequencing. Pancreatic DNA was isolated from pools of R26AID+/KIp48CRE+/KI and control R26AID+/+p48CRE+/KI 20-week-old mice, and then PCR-amplified with specific primers and sequenced as previously described (Perez-Duran et al, 2012). Graphs show cytosine mutation frequency overall (total) or at AGCT hotspots. n = 2. *P-value: Elastase1: 0.0382; Elastase2a: 0.009. C HTM-mediated quantification of γH2AX intensities per nuclei in pancreas explants cultured in vitro for 6 days. Red lines show mean values. Results of two independent experiments are shown. ****P < 0.0001.D Representative images of γH2AX staining in pancreas explants from R26AID+/+p48CRE+/KI (top) or R26AID+/KIp48CRE+/KI mice (bottom) (40× magnification). White arrow points a representative positive cell.
Coi12p is necessary for the loading of scnRNAs to Twi1p in cell lysatesA schematic drawing of the scnRNA loading assays in cell lysates.Figure 4A A schematic drawing of the scnRNA loading assays in cell lysates. The circled samples (B‐i, B‐ii, B‐iii, and B‐iv) correspond to the lanes shown in (B). The boxed samples (C‐i, C‐ii) correspond to the lanes shown in (C). See text for details. B The 27‐nt RNAs that co‐precipitated with Twi1p from lysates with the different conditions shown in (A) were separated on a denaturing gel and detected by autoradiography (top). Precipitated Twi1p was detected by Western blot using an anti‐Twi1p antibody (bottom). C Proteins in the cell lysate before (i) and after (ii) immunodepletion of Coi12p were analyzed by Western blot using anti‐Coi12p (top) and anti‐Twi1p (bottom) antibodies. D Cell lysate from the cells expressing FLAG‐HA‐tagged wild‐type Twi1p (WT) or Twi1p in which the aspartic acid 526 was replaced with asparagine (D526N) at 3 hpm was incubated with radiolabeled double‐stranded 27‐nt RNAs. The Twi1p‐containing complex was immunoprecipitated with an anti‐Twi1p antibody, co‐precipitated RNAs were separated on a native gel, and 27‐nt RNAs were detected by autoradiography. As markers, ssRNA1 and ssRNA2, which were used for producing the radiolabeled double‐stranded 27‐nt RNAs, were mixed in 2:1 (left) or 1:2 (right) ratio, allowed to form double‐stranded RNAs, and were separated in the same native gel.
Figure 5A, B RNF123 interacts with RIG-I/MDA5 CARD. HEK293T cells (1×106) were transfected with expression plasmids for Flag-RNF123 together with HA-RIG-I or its truncations (8 μg each). Co-immunoprecipitation and whole-cell lysates were analyzed as described in the legend of Fig. 3D. C, D RNF123 competes with VISA for RIG-I/MDA5 CARD binding. HEK293T cells (1×106) were transfected with expression plasmids for HA-RIG-I CARD/MDA5 CARD alone or together with Flag-VISA or with Flag-VISA and Flag-RNF123 (8 μg each). Co-immunoprecipitation and whole-cell lysates were analyzed as described in Fig. 3D. E RNF123 does not compete with REUL for RIG-I CARD binding. The methods of transfection and co-immunoprecipitation were as described in Fig. 5C,D. F Knockdown of RNF123 facilitates the endogenous interaction between RIG-I and VISA in 293T cells. 293T cells stably infected with SeV were treated for the indicated times. Cell lysates were immunoprecipitated with anti-VISA antibody. The immunoprecipitates and lysates were analyzed by western blotting with the indicated antibody. G Knockdown of RNF123 facilitates the endogenous interaction between MDA5 and VISA in HeLa cells. HeLa cells were stably infected with EMCV for the indicated times. Co-immunoprecipitation and whole-cell lysates were analyzed as described in (F).
In apoptotic Drp1 knockdown cells, the formation of Bax-rings is not sufficient for full cytochrome c release.Figure 4A In apoptotic Drp1 knockdown U2OS cells, cytochrome c release is delayed. Confocal overview image of an apoptotic Drp1 knockdown cell labelled with antibodies against Tom20 (red), cytochrome c (blue) and Bax (green). Large image: Overlay of all three channels. Small images: Magnifications of the area indicated by the rectangle in the large image. Shown are the individual channels, as indicated. The arrows point to mitochondria that had not released cytochrome c; the asterisks denote mitochondria that had released cytochrome c. Scale bars: 10 µm (overview) and 2 µm (magnifications). B In Drp1 knockdown cells Bax-ring formation and cytochrome c release is not correlated. Three-color images of apoptotic mitochondria of Drp1 knockdown cells. From left to right: Tom22, Bax (recorded in the STED-mode), cytochrome c and an overlay of all three channels. The arrows point to Bax-rings. Scale bar: 1 µm.
Figure 1A Baseline blood counts of granulocytes. One-way ANOVA; n = 8 mice per ZT; *P = 0.0001 ZT5 vs. ZT13. B Flow cytometry quantification of neutrophils in digested hearts. The representative dot plots show the gating strategy for cardiacneutrophils (CD45+Ly6G+CD11b+) at ZT5. One-way ANOVA; n = 5 mice per ZT; *P = 0.0001 ZT5 vs. ZT13. C Representative immunostainings for neutrophils in the myocardium (left ventricle), identified as Ly6G-positive (20x magnification). D Cardiac mRNA expression levels normalized to HPRT. One-way ANOVA; n = 5 mice per ZT; ZT5 vs. ZT17: *P = 0.0064 (CXCL1), *P = 0.0007 (CXCL2), *P = 0.0007 (CXCL5), *P = 0.0001 (ICAM-1), *P = 0.0009 (VCAM-1), *P = 0.0181 (CCL3), *P = 0.0360 (CCL5). E Mean fluorescence intensity (MFI) of CXCR2 expression by neutrophils in blood and heart. One-way ANOVA; n = 3 mice for ZT1, ZT17, ZT21 and n = 5 for ZT5, ZT9, ZT13; ZT5 vs. ZT13: *P = 0.0425 (blood), *P = 0.0078 (heart).
Mec1-S1991 phosphorylation controls Mec1 kinase activity on HU.Figure 7(A) Phosphopeptide abundances (log2 ratio [WT/mec1-S1991A]) in S phase cells treated with 0.2M HU for 60 min. Colored dots are factors with significantly different phosphopeptide fulfilling a combined criteria [WT/mec1-S1991A] >4 (blue) or <4 (red) and p value<0.05 in HU-treated cells (significant phosphopeptides, n= 25 and n=129, respectively). (B) Amongst the Mec1-S1991-dependent phosphopeptides on HU, factors involved in transcription are highlighted. (C) Phosphotargets and subunits with decreased abundance on chromatin identified under HU-induced replicative stress from the chromatome Common complexes are indicated in blue or red. (D) GAL1 and GAL10 mRNA levels measured by RT-qPCR in asynchronous culture in 2% galactose and after 90 min on HU in the indicated strains. Expression is normalized to ACT1. Repression is expressed as a ratio of HU treated/asynchronous cells in percentage. (E) Position relative to the nuclear envelope (Zone 1 of the lacO-tagged GAL1-GAL10 locus in the indicated strains grown either 12h on glucose, or 90 min galactose or galactose + 0.2M HU. The third panel shows the relative retention at the nuclear periphery (Zone 1) on galactose + HU. The number of cells is >300 for each condition.
Unless otherwise stated, all experiments were repeated three times and data shown are representative.figf7(a) Bec-1C. elegans were reconstituted with either WT or mutant GFP-BEC-1. Stable worm lines with Bec-1 rescue were obtained and embryos were stained with anti- PGL-1 antibody. Arrowheads indicate normal PGL-1 staining in germline cells. (b) Quantification of PGL-1 puncta outside germline cells (left panel). Error bars represents s.d. between 3 unique embryos in a representative experiment. Reconstituted Bec-1 (WT and mutant) levels in Bec−/− stable worms were compared by western blotting. Mean value presented. (c) Spectrum of defects in PGL granule degradation in bec-1 mutant rescue embryos. Mutant embryos exhibited either high levels of diffuse PGL-1 staining (middle-left panel; one-third of the embryos), or large punctuate PGL-1 structures in somatic cells (bottom-left panel; two-thirds of the embryos). Both diffuse or punctuate PGL-1 staining in somatic cells have been described in autophagy-deficient embryos. (d) Embryos from the lines described in a were labelled with anti-LGG-1, along with WT and unc-51 worms. Representative embryos at ∼ 100 cell stage are shown. (e) Quantification of LGG-1 per embryo from labelling in d. Error bars generated as in b. Mean value presented. Uncropped images of blots are shown in Supplementary Fig. S4. Scale bar, 10 μm.
NAV3 knockdown enhances invasiveness of mammary cancer cells in vitro and their metastasis in vivo.Figure 3shCTRL- and shNAV3-MDA-MB-231 cells were plated in invasion chambers, and 18 h later, invaded cells were stained. For 3D growth, cells were seeded in Matrigel and photos were taken 5 days later (scale bars, 200 μm and 50 μm).Histograms (means ± SD) of the results presented in (A) (from three experiments). MDA-MB-231 derivatives were plated on coverslips pre-coated with a fluorescent gelatin. Two hours later, cells were probed for actin and TKS5. The number of cells with invadopodia were quantified in three experiments. Shown are means ± SD. Each determination included 80 cells. Scale bar, 10 μm. MDA-MB-231 derivatives were monitored for invasion and proliferation using real-time impedance (xCELLigence). The indicated RFP-expressing MDA-MB-231 derivatives were injected either intravenously (1.5 × 105 per mouse; tail vein), or into the mammary fat pad (2.5 × 106 cells per mouse) of SCID mice. Four weeks later, lungs from the tail vein-injected mice were photographed and examined for RFP signals corresponding to relatively large nodules, which were quantified. Photographs and numbers of the relatively small metastases, as well as tumor size from mice injected into the fat pad, were obtained 8 weeks post-implantation (right panels).
Figure 8Model depicting how physiological signals such as growth and fasting-feeding are integrated at the ER membrane via the Sel1L-Hrd1 ERAD complex in liver to regulate CREBH protein turnover at the ER membrane, and Fgf21 transcription and metabolic states in the body. New findings elucidated by this study are highlighted in red arrows.
Figure 6(A) Primary hippocampal neurons (DIV6+3) were transfected with either VPS4B or an empty vector control together with GFP-RAB11 to visualize recycling endosomes. Neurons were imaged as in Figure 1 to analyze recycling endosome transport. Quantitative analysis of vesicle motility (B) and number (C) from kymographs (n = 4). (D) Primary hippocampal neurons (DIV6+3) were transfected with the indicated combinations of shRNA targeting TDP-43, VPS4B and control together with GFP-RAB11 and imaged as in Figure 1A. Quantitative analysis of vesicle motility (E) and number (F) from kymographs (n=3).
a) LC-MS/MS analysis showing the level of Glc-Cer in control, GBA-PD, isogenic controls and GD iPSC-derived neurons.f5(a) LC-MS/MS analysis showing the level of Glc-Cer in control, GBA-PD, isogenic controls and GD iPSC-derived neurons. Data are represented as mean+s.d.; n=3, *P0.01, One-way ANOVA. (b) Control iPSCs (line C1-1) were differentiated for 65 days and exposed to the GCase inhibitor CBE (25 μM) for 48 h. Representative western blot showing increased levels of α-syn in CBE-treated iPSC-derived neurons compared to untreated neurons. (c,d) Representative western blots showing the protein levels of α-syn in control, GBA-PD, gene-corrected isogenic controls and GD iPSC-derived neurons. (c,d) Representative western blots showing the protein levels of α-syn in control, GBA-PD, gene-corrected isogenic controls and GD iPSC-derived neurons. (e) Optical densities of α-syn bands normalized by the averaged value of GAPDH and expressed as a percentage of the control (CTRL) line value. Data are represented as mean+s.d.; experiments were independently repeated five times. *P0.01, one-way ANOVA. (f) Average α-syn protein levels normalized by the averaged value of GAPDH and expressed as a percentage of the respective isogenic mutant lines. Data are represented as mean+s.d.; experiments were independently repeated five times. *P0.01, Student's t-test.
Figure 4Relative expression of HIV-1 gag in cells infected with Tat-deficient HIV-1pNL4-3 as compared to cells infected with wild type HIV-1 pNL4-3. relative expression of the limiting rate enzyme of the pentose phosphate pathway, i.e. G6PD (B) in HIV-1 infected as compared to mock infected cells. relative expression of genes regulating the thioredoxin and glutathione antioxidant pathways, i.e. TrxR1 (C) and GCLC (D), in HIV-1 infected as compared to mock infected cells.
Figure 4A. Correlation analysis of PTENε protein level with eIF2A or eIF5 mRNA level. The mRNA level of eIF2A and eIF5 in cell lines as listed in Fig 1B was measured by RT-PCR. PTENε protein level in cell lines listed in Fig 1B was measured by evaluating the gray value of corresponding protein bands. PTENε protein level is positively correlated with the eIF2A mRNA level (p < 0.05), while it has no significant correlation with the eIF5 mRNA level (p >0.05). The Pearson correlation test was employed to analysis the correlation between PTENε and eIF2A or eIF5 and data are presented as the mean ± SD based on three independent experiments. B. Reduction of PTENε in response to eIF2A knockdown. Hela cells were infected with lentivirus expressing eIF2A shRNA, eIF5 shRNA, or scramble shRNA separately. The knockdown of eIF2A or eIF5 was validated by western blot (left panel). Lysates of Hela cells were collected and subjected to western blot analysis with antibodies against PTEN, and GAPDH (right panel). C. Overexpression of eIF2A induces the relative expression of PTENε. FLAG-tagged eIF2A or eIF5 were overexpressed in DU145 cells before western blot analysis of PTENε expression with an antibody against PTEN. GAPDH was used as a loading control. D. A palindromic motif 14 bp downstream CUG816 in the 5′UTR of PTEN is evolutionarily conserved. (upper panel) Phylogenetic analysis of the 5′UTR of PTEN mRNA in bonobo (Pan paniscus), killer whale (Orcinus orca), and mouse (Mus musculus). The CUG816 initiation codon of PTENε is highlighted in a blue box and the 14 bp downstream palindromic sequence is highlighted in a red box. The putative hairpin structure shown in the lower panel was predicted by the Mfold web server. Abolition of PTENε expression by disruption of the palindromic motif. (E) Disruption of the CUG816 downstream palindromic motif by mutagenesis. Abolition of PTENε expression by disruption of the palindromic motif. C-terminal FLAG-tagged PTENε expression plasmids with or without disruption of the palindromic motif were introduced into HEK293 cells, followed by immunoblotting with FLAG and GAPDH antibodies (F).
a, Linear regression analysis of nuclear REST levels in PFC neurons and cognitive test scores.f6a, Linear regression analysis of nuclear REST levels in PFC neurons and cognitive test scores. Nuclear REST was imaged by anti-REST immunofluorescence and quantified by MetaMorph. Each point represents an individual case. n = 111 cases (59 females, 52 males), age range 71-90 years. b, Nuclear REST levels are inversely correlated with Braak stage (extent of neurofibrillary tangle formation). c, Nuclear REST levels in PFC from cases with neuropathological AD (moderate/frequent plaques by CERAD score and intermediate/high likelihood AD by NIA-Reagan criteria) that had mild or no cognitive impairment (NCI/MCI), or AD dementia. Values are the mean ± s.e.m. *P 0.01 by Student's unpaired t-test; NCI/MCI n = 30, AD dementia n = 21. d, Nuclear REST levels in ageing neurons correlate positively with longevity. REST levels were quantified by FACS analysis of isolated PFC neuronal nuclei in 61 individuals without AD (age range 67-104 years).