EMBO/sd-character-level-ner huggingface dataset:
Figure 1A BAK-6H permeabilising activity is exacerbated by cBID. BAK-6H (mBAK∆C21-6H) was loaded at the indicated concentrations onto Ni-NTA liposomes (5 μM) prior to the addition of cBID. The release of carboxyfluorescein was measured over time. Results representative of at least 3 independent experiments. B BAK-6H oligomerises on liposomes induced by cBID. Liposomes were incubated with BAK-6H (150 nM) and the indicated concentration of cBID or a BID BH3 peptide for 60 min. Liposomes were solubilised in digitonin and BAK oligomers were analysed by BN-PAGE. In parallel, samples were tested at endpoint for liposome permeabilisation based on the release of flurorescent dextran, normalised to total fluorescence of detergent permeabilised liposomes. Results representative of 3 independent experiments. C BAK-6H oligomerisation on liposomes is blocked by BCL-XL. Liposomes were incubated with BAK-6H (150 nM) together with cBID (WT, 90 nM) or cBID M97A (90 nM) that has reduced affinity for BCL-XL, in the presence or absence of BCL-XL for 60 mins prior to analysis of BAK oligomerisation by BN-PAGE. Results representative of 2 independent experiments. Liposome permeabilisation was monitored with the indicated combinations of cBID (90 nM), BAK-6H (150 nM) and BCL-XL (1 μM). Data is presented as mean of 2 independent experiments with percentage release relative to detergent-lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50% of detergent-lysis maximum. D BAK-6H oligomerisation on liposomes involves its BH3 domain. Liposomes were incubated with BAK-6H (150 nM) together with cBID (90 nM). Antibody recognising the BAK BH3 domain (4B5) or α1-α2 loop (7D10), as shown on the structure of BAK (PDB:2IMS), were added (2.4 μg/μl) either prior to (pre) or after (post) incubation with cBID for 60 mins. BAK:Ab complex is indicated (arrowhead). 7D10 which recognises the activated form of mBAK (Iyer et al., 2016), bound to and gel-shifted BAK when added either before or after activation with cBID. In contrast, 4B5 failed to appreciably gel-shift inactive BAK (lanes 3 and 5) or BAK that was already activated (lane 6), but gel-shifted BAK when present during cBID activation (lane 4). Liposome permeabilisation was assessed after 20 min incubation with cBID (90 nM), BAK-6H (150 nM) and antibodies 4B5 (2.5 μg) or 7D10 (2.5 μg). Data is presented as mean of 2 independent experiments with percentage release relative to detergent-lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50% of detergent-lysis maximum. E HDX-MS reveals structural constraints of BAK on a membrane. BAK-6H (150 nM) on liposomes was deuterated for 0, 10s, 60s or 600s and the incorporation of deuterium was assessed by mass spectrometry. Deuteration is expressed relative to a theoretical maximum. BH3 domain (orange), BH4 domain (blue) and hydrophobic surface groove comprising α helices 3-5 (green) are shown on the structure of BAK (PDB:2IMS). F Differential deuteration of BAK in a membrane. Relative deuteration is mapped onto the structure of inactive monomeric BAK
Figure 5A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA were subjected to wound-healing (A) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance; One-way ANOVA. Scale bar in (A): 200 μm. A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA Lysates from these cells were also analyzed by western blot (B). Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA were subjected to transwell migration (C) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance; One-way ANOVA. Scale bars in (C : 100 μm. A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA were subjected to transwell invasion (D) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance; One-way ANOVA. Scale bars 100 μm. A549 cells expressing either control shRNA or WDR63 shRNA#1 were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell migration (E) The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm. A549 cells expressing either control shRNA or WDR63 shRNA#1 were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Lysates from these cells were also analyzed by western blot (F). Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. A549 cells expressing either control shRNA or WDR63 shRNA#1 were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell invasion (G) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.
Figure 4Left panel: Representative Western blots illustrating the expression of SMC6, SMC5 and FANCD2 in protein extracts from HeLa cells 48 hours after transfection with the indicated siRNAs. Right panel: Relative expression of SMC5, SMC6 and FANCD2. We performed individual experiments for each cell line with technical replicates. In each individual experiment, we first normalized SMC5, SMC6 or FANCD2 expression to that of Vinculin (internal control) and calculated its relative level in each cell line in relation to the target protein/Vinculin ratio of the siLacZ transfected cells, which was set as one in each experiment. Data present the mean+/-S.E.M. and were analyzed by unpaired t-test. SMC5 in siSMC6: n=7 and p=0.0002; SMC5 in siFANCD2: n=7 and p=0.0016; SMC6 in siSMC5: n=8 and p<0.0001; SMC6 in siFANCD2: n=24 and p<0.0001; FANCD2 in siSMC6: n=30 and p<0.0001; FANCD2 in siSMC5: n=10 and p=0.0008. Top. Representative Western blots illustrating the expression of SMC6, FANCJ and FANCD2 in protein extracts from HeLa cells 48 hours after transfection with the indicated siRNAs. Bottom. Survival curves of cells transfected with indicated siRNA and treated with different concentrations of mitomycin C (MMC). Data represents the means and SEM (standard error of the mean) of 3 independent experiments.
nNOS membrane dissociation, FoxO3A accumulation in myonuclei and upregulation of autophagy‐related genes as consequences of α1S subunit loss.Figure 3(A) mRNA from TAΔDHPR and TACtrl tissues were extracted and nNOS expression was quantified by qRT-PCR. Results are expressed as mean±s.e.m., **P0.001, n=4. (B) Transversal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐nNOS (red), anti‐laminin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm. (C) The tissues from extracts were analysed by western blot with FoxO3a‐P antibody and normalized with α‐actin antibody. Graph depicts mean±s.e.m. of relative expression of FoxO3a‐P determined by densitometry and nomalized to the α‐actin expression for each muscle, *P⩽0.005, n=3. (D) Longitudinal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐FoxO3a (red), anti‐laminin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm. (E) mRNA from TAΔDHPR and TACtrl tissues were extracted and regulation of autophagy genes expression was followed by qRT-PCR. Bnip3, CathepsinL, LC3 and PI3KIII expression (noted in red) were significantly increased in TAΔDHPR compared with the controlateral TACtrl, **P0.001, n=4.
a) dRagA positively regulates wing compartment size.figf4(a) dRagA positively regulates wing compartment size. Wild-type or mutant dRagA transgenes were expressed in posterior compartments with the en-GAL4 driver. The ratios of representative posterior to anterior compartment areas are shown. Posterior compartment area was significantly increased in response to dRagAQ61L expression and decreased in response to dRagAT61N expression. Data are mean ± s.d., *P = 7.32 × 10−4 (n = 7), **P = 6.18 × 10−4 (n = 12), Student's two-tailed t-test, (n is the number of adult wings analysed). (b) dRagA positively regulates wing cell size. The average area of posterior compartment cells from en-GAL4 UAS-dRagA adult wings is shown. Cells expressing dRagAQ61L are significantly larger and dRagAT61N-expressing cells are smaller than controls. Data are mean ± s.d., *P = 1.15 × 10−3 (n = 7), **P = 0.025 (n = 12), Student's 2-tailed t-test (n represents number of adult wings analysed). (c, d) dRag GTPases positively regulate larval fat body cell size. (c) Cell area of clonally-induced dRagA-expressing cells or dRagC homozygous mutant cells relative to neighbouring wild-type control cells is shown. Cell area was determined from phalloidin-stained fixed fat body samples from fed or 48 h starved larvae. Expression of dRagAWT or dRagAQ61L significantly increased relative cell area under starvation but not fed conditions. Cells expressing dRagAT61N and dRagC loss-of-function cells were significantly smaller than control cells only under nutrient replete conditions. Data are means ± s.d., *P = 2.04 × 10−3 (n = 5), **P = 2.94 × 10−6 (n = 14), ***P = 3.79 × 10−7 (n = 14), ****P = 1.36 × 10−5 (n = 30), Student's two-tailed t-test. (d) Representative examples of fat body cells with altered dRagA activity. Cells expressing dRagA transgenes are marked by the expression of GFP in the left and middle panels, and dRagC homozygous mutant cells are marked by absence of GFP in the right panel. Scale bar represents 50 μm.
Figure 1A. The three normal RNA-binding FET proteins consist of N-terminal repetitive and structurally disordered domains (NTD: N-terminal domain), central RNA-binding domains (RRM: RNA recognition motifs), zinc finger domains (ZF) and degenerated repeat regions (RGG: RGG repeat regions). The GST-tagged FET-NTD baits shown were used for pulldown experiments and represent the shortest parts commonly present in the FET fusion oncoproteins. The FET binding motif is a conserved sequence required for complex formation between the three FET proteins. Amino acid numbers are indicated. B. Schematic illustration of a representative FET fusion protein (FUS-DDIT3 Type II) and its parental proteins. DBD: DNA-binding domain, LZ: leucine zipper domain. Type I and Type II show locations of the two most common MLS fusion breakpoints in FUS. C. Coomassie staining of SDS-PAGE-separated pulldown samples with/without cell extracts (C.E) using GST-tagged FET-NTDs as baits. Sepharose with bound GST was included as control. Background from recombinant protein baits (shown by white arrowheads) as well as smaller partial recombinant products is visualized in the left panel (C.E -). Several high-molecular weight proteins are retained by the FET-NTDs (C.E +). Black arrowheads indicate protein bands and gel parts analyzed by mass spectrometry D. Immunoblot analysis (IB) of pulldown samples with FET-NTD baits with/without cell extracts (C.E). Antibodies against ARID1A, BRG1 and BAF60A were used for detection of SWI/SNF components. Input samples diluted 1:5 were included as a control. E. In situ proximity ligation assays (PLA) using antibodies against BRG1, ARID1A, DDIT3 and C-terminal parts of normal FUS show protein complexes containing FUS/BRG1, FUS/ARID1A, FUS-DDIT3/BRG1 and FUS-DDIT3/ARID1A as red fluorescent spots in nuclei of MLS cell lines 2645-94 and 402-91. C-terminal parts of FUS are not present in the FUS-DDIT3 fusion protein and normal DDIT3 is not expressed in these cell lines. Merged images also include DAPI nuclear counterstain in blue. Combinations of primary antibodies are used to detect interactions (left panel). In control experiments (right panel), one primary antibody is omitted to evaluate the background fluorescent signals. Scale bars = 10 μm.
Figure 9 Figure 9. Representation of the observed changes in Alu RNA processing in AD. In no AD brains, basal Alu RNA processing levels maintain control of gene transcription through regulation of RNA Pol II. In contrast, in AD pathology, various, yet unknown, factors lead to an increase in Alu RNA processing associated with transcriptome-wide de-regulation of gene expression.
Figure 2(A) Experimental flow of sample generation for quantitative mass spectrometry. (B) Volcano plot showing the most enriched/decreased proteins in the mitochondrial proteome of pex19Δ versus pex19Δmsp1Δ strains as detected by quantitative mass spectrometry (n=7, biological replicates; n=2, technical replicates). Effect sizes were calculated for Cohen's d A protein's relevance was determined using a p-value threshold of < 0.05 with an absolute effect size threshold of 1 or greater (large effect). Gray dots represent all measured proteins and red dots highlight peroxisomal-associated proteins. (C) Heatmap of peroxin proteins from pex19Δ and pex19Δmsp1Δ stains as detected by quantitative mass spectrometry (n=5). Protein classes are indicated in the row labels. Values were z-score normalized by protein. (D) The indicated yeast strains expressing PEX13-RFP and mitochondria-targeted green ﬂuorescent protein (mGFP) were grown to mid-log phase and visualized by ﬂuorescence microscopy. Representative images are shown, scale bar, 2 μm. DIC, differential interference contrast. (E) The indicated yeast strains expressing PEX11-RFP and mitochondria-targeted green ﬂuorescent protein (mGFP) were grown to mid-log phase and visualized by ﬂuorescence microscopy. Representative images with enhanced contrast and brightness are shown, scale bar, 2 μm. DIC, differential interference contrast. (F) Total yeast cell lysate, post mitochondrial supernatant, and HistodenzTM-purified mitochondria from the indicated strain (wild type, msp1Δ, pex19Δ, pex19Δmsp1Δ, pex3Δ, and pex3Δmsp1Δ) expressing Pex13-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for Pex13 (α-V5), cytochrome c (α-cyt c), and HSP70 (α-hsp70). (G) Total yeast cell lysate, post mitochondrial supernatant, and HistodenzTM-purified mitochondria from the indicated strain (wild type, msp1Δ, pex19Δ, pex19Δmsp1Δ, pex3Δ, and pex3Δmsp1Δ) expressing Pex11-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for Pex11 (α-V5) and porin (α-porin), cytochrome c (α-cyt c), and HSP70 (α-hsp70).
Figure 3(A) A ~44 kD protein band specifically pulled down by GST-WDR63 from A549 cell lysates was identified as Arp2 by mass spectrometry analysis. The Arp2 peptide sequences obtained by MS are shown. (B) Lysates from HEK293T cells expressing HA-Arp2 alone or together with Flag-WDR63 were immunoprecipitated by anti-Flag antibody, followed by western blot analysis. Data shown represent three independent experiments. (C) Lysates from HEK293T cells expressing GFP-WDR63 alone or together with Flag-Arp2 were immunoprecipitated by anti-Flag antibody, followed by western blot analysis. Data shown represent three independent experiments. (D) Lysates from A549 cells were immunoprecipitated with control IgG or anti-WDR63 antibody, followed by western blot analysis. Data shown represent three independent experiments. (E) Purified GST or GST-Arp2 proteins immobilized on glutathione beads were incubated with purified Flag-WDR63. Input and bead-bound proteins were analyzed by western blot. Data shown represent three independent experiments. (F) Purified GST or GST-WDR63 proteins immobilized on glutathione beads were incubated with purified Arp2. Input and bead-bound proteins were analyzed by western blot. Data shown represent three independent experiments. (G) Lysates from HEK293T cells expressing GFP-WDR63 alone or together with the indicated Flag-tagged each single subunit of the Arp2/3 complex were immunoprecipitated with anti-Flag antibody, followed by western blot analysis. Data shown represent three independent experiments. (H) Purified Flag-tagged full-length or the indicated truncated WDR63 proteins immobilized on anti-Flag M2 beads were incubated with the purified Arp2/3 complex (Cytoskeleton, Inc.) in vitro. Input and beads-bound proteins were analyzed by western blot. Data shown represent three independent experiments.
Figure 3Figure 3. 3D replication profile.(A) Comparison of replication profiles of the synchronized populations used for the analysis displayed in Fig 2G. The read coverage of raw Hi-C libraries reflects the replication progression throughout S phase, plotted along the 16 chromosomes of the yeast genome (top axis; blue curve). The replication timing obtained in this study is highly similar to the one from McCune et al., 2008 (yellow curve). Figure 3. 3D replication profile. (B) Superposition of three 3D representations of chromosomes in early replication (I, II, III). The color scale indicates the replication timing. Centromeres and telomeres are highlighted. Different view of the structure are presented. Figure 3. 3D replication profile. (C) Pattern of the replication profile for each of the chromosomal arm. The color code reflects the timing of replication.
Figure 4(A) ARSA overexpression detected by IHC in cortical and subcortical regions of the injected and contralateral hemispheres of NHP S2.3. (B) Physiological ARSA expression in cortical and subcortical regions of the injected hemisphere in LV.GFP-injected NHP P1. Scale bar, 100 µm. Insets show ARSA-expressing cells at higher magnification. Note the perinuclear accumulation of ARSA protein. (C) Quantification of immunopositive signal in brain slices 3 to 6 (n= 8 blocks/slice). Data are expressed as fold increase to mean immunopositive signal measured in matched brain regions of NHP P1 (physiological). Two-way ANOVA and Bonferroni post-tests, *p<0.05, **p<0.01, ***p<0.001 vs physiological.
Figure 1(A) Schematic diagram of establishment of KLF4 loxp/loxp coupled AAV7-Cre inducible mouse. (B) Genotyping of KLF4loxp/loxp coupled AAV-Cre inducible mouse. 2x1011 particles of AAV7-Cre-mCherry or AAV7-mCherry were intraperitoneally injected into KLF4 loxp/loxp mice (6~8 weeks). Five weeks later, mice tails were cut and collected for DNA extraction and PCR analysis. PCR results were analyzed by 1% agarose gel. Validation of inducible knockout of KLF4 in mouse intestine. Five weeks after injection of AAV7-Cre-mCherry, mouse intestine was removed and followed by the preparation of tissue section. The KLF4 expression in the intestine was then detected by Western blot Validation of inducible knockout of KLF4 in mouse intestine. Five weeks after injection of AAV7-Cre-mCherry, mouse intestine was removed and followed by the preparation of tissue section. The KLF4 expression in the intestine was then detected by immunohistochemistry (E) Kaplan-Meier survival curves of KLF4 loxp/loxp mice with intraperitoneal injection of AAV7-Cre-mCherry or AAV7-mCherry followed by the treatment with 8-Gy (total-body) γ-irradiation 5-weeks after then. AAV7-mCherry, n=10 per group; AAV7-Cre-mCherry, n=12 per group. p = 0.0023, log-rank test. (F) Histological analysis of intestinal epithelium of KLF4 loxp/loxp mice with AAV7-mCherry or AAV7-Cre-mCherry intraperitoneal injection followed by 8-Gy (total-body) γ-irradiation 5-weeks after then. Tissues were collected from the sham mice and mice at different time after exposure to γ-irradiation. Scale bars, 100 μm. (G-H) Immunofluorescent staining of γ-H2AX, 53BP1 and Cleaved Caspase-3 in the intestinal epithelium of KLF4 loxp/loxp mice with injection of AAV7-mCherry or AAV7-Cre-mCherry followed by treatment of γ-irradiation. Tissues were collected from sham mice and mice at different time after exposure to γ-irradiation and then staining with indicated antibodies. (G) Quantification of γ-H2AX, 53BP1 and active Caspase-3 -positive cells based on the Immunofluorescent staining results presented in H. Data are mean ± SEM; n=4 per group; p = 0.0023 (r-H2AX), p = 7.9x10-4 (53BP1) p = 5.5x10-3 (Active Caspase-3). one-way ANOVA was used for the statistical analysis. Scale bars, 60 μm.
Figure 1Senescence-associated β-galactosidase (SA-β-gal) staining in A549, HAFF, HepG2 and H1299 cells after 48h transduction with either negative control lentiviruses (shCtrl) or those targeting GUARDIN (shGUARDIN). The graph shows percentage of SA-β-gal-positive cells. Senescence-associated heterochromatin foci formation (SAHF) in A549 cells with either shCtrl or shGUARDIN. Representative IF staining with H3K9me3 antibodies (green) and nuclear Hoechst counterstaining (blue). Secretion of SASP factors IL-6 and IL-8 in A549 cells with either shCtrl or shGUARDIN. Western blotting for p21, p16 and p15 in HepG2 cells after 48h transduction with shCtrl or shGUARDIN-1, -2, respectively (left). Knockdown efficiencies analyzed by qPCR (right). SA-β-gal staining in A549 cells after 24h transduction with either shCtrl or shGUARDIN in combination with p21 shRNA (shp21). The graph shows percentage of SA-β-gal-positive cells. qPCR measurement of p21 mRNA in A549, HepG2, HAFF and H1299 cells with either GUARDIN shRNA knockdown (top) or GUARDIN overexpression (bottom). Western blotting for p53 and p21 in A549, HepG2, HAFF and H1299 cells with either GUARDIN shRNA knockdown (top) or GUARDIN overexpression (bottom). qPCR assays of NOXA, TIGAR, BAX and p21 mRNA levels in A549 cells with either shCtrl or shGUARDIN.
Fig.Figure 6A, B, Biolayer interferometry analysis with Apo-PGP A and holo-PGP B. Proteins were immobilized at a streptavidin sensor tip. Free binding sites were blocked with biotin. Association and dissociated kinetics were recorded at the indicated HoxA concentrations. Kd values were calculated with the manufacturers software using a global fit for both experiment