EMBO/sd-character-level-ner huggingface dataset:
Mitochondrial stress does not impair the DNA-damage checkpoint.Figure 3Time course quantification of apoptotic corpses relative to the total number of meitic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 (A and left untreated or exposed to 1200 J/m2 UVB. Apoptotic cells were counted 12, 24 and 36 hours after exposure Data information: Data are presented as mean ± SEM, N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons Time course quantification of apoptotic corpses relative to the total number of meitic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) o isp-1 (B), and left untreated or exposed to 1200 J/m2 UVB. Apoptotic cells were counted 12, 24 and 36 hours after exposure Data information: Data are presented as mean ± SEM, N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOV Tukey"s multiple comparisons Real Time PCR gene expression analysis of egl- genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated with gamma radiatio Data information: Data are presented a mean ± SE N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons Real Time PCR gene expression analysis of egl- genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit UV Data information: Data are presented as mean ± SE N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons Real Time PCR gene expression analysis o ced-1 genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated with gamma radiatio Data information: Data are presented as mean ± SE N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons Real Time PCR gene expression analysis o ced-1 genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treate with UV Data information: Data are presented as mean ± SE N= F). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s mu comparisons test
The Wnt/β‐catenin/TCF pathway controls p62 expression.Figure 4(A) Relative p62/SQSTM1 mRNA levels by quantitative real‐time‐PCR (qRT-PCR) increased following β‐catenin knockdown in HT29 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). β‐Catenin knockdown was confirmed by western blotting. (B) Relative p62 mRNA levels by qRT-PCR decreased with β‐cateninS33Yoverexpression after 24 h of starvation in HCT116 β‐cateninWT/− cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). β‐CateninS33Y overexpression was confirmed by western blotting. (C) The increase in p62 protein expression induced by β‐catenin siRNA was attenuated in HT29 cells treated with 10 μg/μl cycloheximide and starvation for 8 and 24 h compared to vehicle control. A complementary experiment using the transcription inhibitor actinomycin D is shown in Supplementary Figure S1C. (D) Overexpression of Fzd6 (green, left panel) increased p62 (red, right panel) protein expression in HEK293T cells. As an internal control, cells not overexpressing Fzd6 are shown in the field of view. (E) Doxycycline induction (1 μg/ml) of DNTCF4 increased p62 and LC3‐II protein expression in doxycycline‐inducible DNTCF4 LS174T‐L8 cells. LGR5 downregulation by DNTCF4 confirmed inhibition of β‐catenin/TCF4 signalling. (F) Relative p62 mRNA levels (48 h) by qRT-PCR increased following doxycycline induction of DNTCF4 in LS174T‐L8 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). (G) Upper panel: Wnt3a (200 ng/ml) treatment of HCT116 β‐cateninWT/− cells (24 h) decreased LC3‐II and p62 protein expression by western blotting. Lower panel: quantification by densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of three independent experiments).Source data for this figure is available on the online supplementary information page.
Figure 6A Comparison of the cryo-EM structure of H2A.Z.2.2-NCP with the structure of canonical nucleosome (PDB: 3LZ0) in disc view. Histone octamers containing H2A.Z.2.2 and canonical H2A are superimposed for comparison. The 125 bp DNA of Z.2.2-NCP in green color is shown in surface mode. The 145 bp DNA of canonical nucleosome is colored in grey. Histones H2A.B, H2B, H3 and DNA at SHL 6 through SHL 7 are highlighted to show the structural differences between two nucleosomes. The Z.2.2-NCP histones are colored in orange, warmpink, and cyan. Their counterparts in canonical nucleosome are colored in yellow, red, and blue. Histone H4 is colored in grey. B Close-up view of the structures of Z.2.2 C-terminal regions (top) and H2A C-terminal regions (bottom). Black and white arrows indicate last residue at the C-terminus of Z.2.2 (G106) and H2A (K118) that are observable in the structures. C Comparison of the cryo-EM structure of Z.2.2-NCP with the crystal structure of canonical nucleosome (PDB: 3LZ0) in gyre view. Arrows indicate the direction of Z.2.2-NCP DNA movement during nucleosome gaping transition. H2B N-tail refers to H2B residues 26-32 which are exclusively observed in the canonical nucleosome structure. Histones H2B and DNA in Z.2.2-NCP are colored in warmpink and green, their counterparts in canonical nucleosome are colored in red and grey. D SDS-PAGE analysis of SWR1 complex purified from S. cerevisiae. E Effect of H2A.Z.2.2 ROF on facilitating histone replacement. Incorporations of yeast H2A.Z-H2B dimers containing either the wild type H2A.Z or Z.2.2-like H2A.Z were analyzed by SWR1-catalyzed histone replacement assay. H2A.Z and Z.2.2-like H2A.Z incorporated into nucleosome were resolved by 6% Native-PAGE and detected by SYBR Green staining and western blotting.
Schematic model of microautophagic degradation of peroxisomes in P.Figure 1Schematic model of microautophagic degradation of peroxisomes in P. pastoris. Microautophagic degradation of peroxisomes is dissected into four stages (see text) depending on the morphology and the appearance and disappearance of intermediates in the process. Mutations in specific PAG genes terminate this process at the stages indicated.
Figure 2(A) Alkaline carbonate extraction of mitochondria from control fibroblasts. Immunoblot analysis shows that SLC25A46 is an integral membrane protein. SDHA (soluble, membrane-associated protein) and MFN2 (integral inner membrane protein) were used as controls. (B) Proteinase K digestion assay on mitochondria from control fibroblasts. Mitochondria were exposed to an increasing concentration of proteinase K to determine the sub-mitochondrial localisation of SLC25A46. SLC25A46 behaves as an outer membrane protein. MFN2 was used as a control for outer membrane protein, AIFM1 for protein present in the inter-membrane space, HCCS and SCO1 for inner membrane proteins and MRPL14 for matrix protein. (C) Immunoblot analysis of mitochondria from the same samples as in (D), for expression of SLC25A46 and known molecular components of the fusion-fission machinery. SDHA was used as a loading control. (D) Immunofluorescence microscopy analysis of the mitochondrial network. Control and subject fibroblasts, overexpressing an EGFP-tagged version of the wild-type SLC25A46 protein (green) and control fibroblasts treated with an siRNA against SLC25A46 were stained with the mitochondrial marker TOMM20 (red) and the nuclear marker DAPI (blue). Scale bars: 10 µm. (E) Quantification of the effect of SLC25A46 on the mitochondrial network shape. In each experimental condition described in (D), 150 cells were analysed and the mitochondrial network organisation was classified as intermediate, fused or fragmented, in three independent experiments. Error bars represent mean +/- SEM and p-values were calculated using a t-test: Control vs patient, p=1.10x10-4. Control vs siRNA SLC25A46, p=1.16x10-4. Subject vs subject+SLC25A46-GFP, p=2.06x10-5.
Figure 4Active EAE was induced by subcutaneous immunization with CFA/MOG35-55. Cells were isolated from the CNS of EAE animals at different stages of the disease (pre-onset [day 8-9], peak [day 14-17], chronic [day 22-27]; data from at least two independent experiments). Representative samples are shown, which were analyzed by flow cytometry for the expression of the markers CD11c, CD11b and Ly6C/G (pre-gated on lympho-monocytic cells by FSC/SSC; IAb+ expression was confirmed for CD11c+ cells in separate stainings).(A) In the CNS, there was a strong increase of CD11c+ proportions over the different phases of the disease. The composition of CD11c+ subpopulations changed over time, starting with a preponderance of CD11c+Ly6C/G- conventional DCs before onset of EAE. In the peak and chronic phase there was a continuous increase in the proportion of monocyte-derived CD11c+ cells.(B) For the CNS, the quantification of the absolute numbers shows a strong increase in CD11c+ cells during the course of EAE.(C) In the CNS, the proportions of the subpopulations show an increasing proportion of monocyte-derived CD11c+ cells.
ATPase is required for chromatin extraction and disassembly of ubiquitylated CMG helicase in mouse ES cells.Figure 2(A) Cells were treated with 5 µM CB-5083 as indicated (p97i = p97 inhibitor). Extracts were then incubated with IgG beads to isolate the GINS complex via TAP-SLD5 and the indicated factors were monitored by immunoblotting. Accumulation of the CMG helicase upon inhibition of p97 was reflected by the increased association of GINS with CDC45 and MCM2-7. (B) Immunostaining of HP1 protein in fixed mouse ES cells that were stained with Hoechst to reveal condensed patches of heterochromatic DNA (examples marked by arrows; colocalization of HP1 and condensed DNA in 83 % of cells, n = 103). (C) Time-lapse analysis of mouse ES cells expressing GFP-SLD5 and mCherry-PCNA Arrows indicate co-localisation on heterochromatic patches, which was observed in 14 % asynchronous cells (n = 238). GFP-SLD5 appeared and disappeared with similar kinetics to mCherry-PCNA (100 % cells, n = 101). (D) GFP-SLD5 mouse ES cells were treated with p97i as indicated and then fixed and stained with Hoechst. Arrows indicate accumulation of GFP-SLD5 on constitutive heterochromatin (53 % cells, n = 125). (E) Immunofluorescence of the replisome components CLASPIN (i) and CTF4 (ii) in GFP-SLD5 cells treated with p97i. Arrows indicate co-localisation of GFP-SLD5 patches with CLASPIN (95 % cells, n = 107) or CTF4 (91 % cells, n = 45). Scale bars in all panels correspond to 10 µm.
Figure 6A Representative water-maze pathway traces of the last four trials of day 3. Trial 1 is shown in red, trial 2 in green, trial 3 in blue and trial 4 in black.B Morris water maze (MWM) analysis of acquisition performance of male (left) and female (right) mice represented as the mean distance (cm) that mice traveled to find the submerged platform plotted per day (mean s.e.m.) Symbols represent statistically significant difference within the same genotype (*AD ASO-C vs AD ASO-21 and #WT ASO-C vs WT ASO-21) or between groups with same treatment (ΔAD ASO-C vs WT ASO-C and ^AD ASO-21 vs WT ASO-21) on the corresponding day (two-way repeated-measures ANOVA with Tukey multiple comparisons test; **p<0.01, ^p<0.05, ΔΔΔΔp<0.0001). See source data for statistical results.C Morris water maze analysis integrated distance (Zhang et al, 2014) (area under the curve, AUC) for male (left) and female (right) WT and AD mice treated with control ASO (ASO-C) or ASO-21. (mean s.e.m.; one-way ANOVA, Tukey post-test.D,E Correlation analysis of performance on day 3 of the MWM with exon 19 skipping among all groups of mice (D) or between AD mice treated with ASO-C or ASO-21 (E). Male and female mice are indicated. Pearson correlation coefficient (r), covariance (σ) and two-tailed p-value are shown.
Figure 3Relative mRNA levels of KDM6A, nephrin and WT-1, normalized to β-actin, expressed in kidney glomeruli of normal and diabetic mice at 4, 8 and 12 weeks after diabetic induction. *Significant differences (P < 0.05) compared with normal controls (Wilcoxon two-sample test; n =8 each). B Western blot analysis of nephrin, KDM6A and H3K27me3 in kidney glomeruli of normal and diabetic mice at different time points. *P < 0.05 versus normal controls for the indicated time points (Wilcoxon two-sample test; n = 3). C Changes in body weights and levels of blood glucose in normal, diabetic and GSK-J4-treated diabetic mice. As noted, GSK-J4 treatment did not significantly affect body weights or blood glucose levels in diabetic mice during the experimental period of 12 weeks (Wilcoxon two-sample test; n = 8). D Levels of urinary protein excretion, weights of kidney, and levels of HbA1c in normal, diabetic and GSK-J4-treated diabetic mice. Urinary total protein excretion, kidney weight and levels of HbA1c were measured at 12 weeks after diabetic induction. The mean relative kidney weight (%) shown in the study is determined as the percent of kidneys out of total body weight, and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin (% HbA1c; DCCT unit). *P < 0.05 versus normal controls, #P < 0.05 versus untreated diabetic mice (Parametric ANOVA and a Bonferroni post hoc test; n = 8). E Western blot analysis of KDM6A, nephrin, WT-1 and H3K27me3 expressed in kidney glomeruli of normal, diabetic and GSK-J4-treated diabetic mice at 12 weeks after diabetic induction. *P < 0.05 versus normal controls, #P < 0.05 versus untreated diabetic mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3). F Immunofluorescence analysis of KDM6A, nephrin and WT-1 in kidney sections from normal, diabetic, and GSK-J4-treated diabetic mice. Green: nephrin or WT-1; Red: KDM6A; Blue: DAPI. Scale bars, 20 μm. *P < 0.05 versus normal controls, #P < 0.05 versus untreated diabetic mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3). G Immunofluorescence staining of F-actin and KDM6A in primary podocytes isolated from normal, diabetic, and GST-J4-treated diabetic mice. Green: F-actin; Red: KDM6A; Blue: DAPI. Scale bars, 20 μm. Presented experiments were performed at least three times independently.
Several key residues in the pore of the PC1/PC2 channel may play roles in ion conductance.Figure 8Structure of the PC1/PC2 complex showing the side view of S10-S11 from the PC1 subunit and S5-S6 from one PC2 subunit in the cryo-EM structure of PC1/PC2 (PDB code 6A70) [42]. Side chains of PC2-L677A and N681 are shown in red and N674 in cyan. Side chains of amino acids on S11 of PC1 at this region were also shown in purple for the three positively-charged amino acids and in green for others. In PC1/PC2 complex, no bulky side chain from PC1 lines up with PC2-L677A and N681 to form the restriction at this position, while R4090 of PC1 and N674 of PC2 narrow down the channel pore in the middle.
Figure 5(A) Schematic representation of the annotated RefSeq gene models of LIMT (NR_002809; chromosome 12q24.31) and the neighboring protein-coding genes (RHOF and SETD1B). Arrows denote direction of transcription (note that LIMT resides on the minus strand). Also shown is the distribution of evolutionary conservation along the sequence of LIMT across 100 vertebrate species. Note that the 5' region of LIMT is highly conserved (green rectangle). Promoter-associated histone methylation (H3K4Me3) and acetylation (H3K27Ac3) were obtained in MCF7 and MCF10A cells, respectively (see Methods). Acetylation signals were obtained for MCF10A cells stimulated with EGF for 20 minutes (green), or un-stimulated (black; overlaying histogram). Note that stimulation with EGF caused a decrease in acetylation at the promoter region (red rectangle). (B) Expression of LIMT was determined in a panel of 44 cancer and non-cancer cell lines using real-time qPCR. Expression levels were normalized to the GAPDH transcript and presented in arbitrary units. N, non-cancer cell line.
TLN mediates phagocytic uptake of microbeads in hippocampal neurons.Figure 9.TLN mediates phagocytic uptake of microbeads in hippocampal neurons. (A) Microbeads align along neurites of hippocampal neurons as demonstrated by phase contrast (DIC). Already after 4 h, many beads stain positive for TLN (arrowheads, top). At 48 h (bottom), most TLN immunoreactivity was associated with microbeads at the expense of its typical plasma membrane staining. *, cell body. Note that microbeads were not found associated with preexisting TLN accumulations (asterisk in top panels, 4 h). (B) Microbeads (24 h) accumulate TLN and actin, as shown by phalloidin-Alexa 568. An overview of the neuron is given in each top left inset, next to two detailed areas (white boxes). The differential interference contrast (DIC) clearly demonstrates a complete colocalization of microbeads with TLN and phalloidin (arrowheads). TOPRO-3 (blue) marks the nucleus (see also Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200406060/DC1). (C) Similar as in B, but immunostained for TLN and endogenous PIP2. The overview demonstrates the overall recruitment of PIP2 to microbeads (arrows). The parallel insets show a detailed area (white box) with PIP2 being colocalized with TLN on individual microbeads (arrowheads). Note that PIP2 occasionally colocalizes with TLN at the plasma membrane (asterisk). Bars (A-C), 10 μm.
Figure 1(A) p53-dependent dephosphorylation of Ulk1 at Ser637. p53+/+ and p53−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells assessed by propidium iodide (PI) staining. α-Tubulin was used as a loading control. A semiquantitative analysis of protein expression is shown in Fig. EV1. (B, C) PPM1D-dependent dephosphorylation of Ulk1 at Ser637 and the induction of autophagy. PPM1D+/+MEFs and PPM1D−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells. (B, C) PPM1D-dependent dephosphorylation of Ulk1 at Ser637 and the induction of autophagy. PPM1D+/+MEFs and PPM1D−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells. (C) Semiquantitative analysis of protein expression is shown (n = 3; mean ± SD). *p < 0.05, **p < 0.01, NS: not significant. (D-I) Suppression of etoposide-induced autophagy in PPM1D−/−MEFs. (D, E) The indicated MEFs were treated with etoposide (10 µM) for 6 hr and then immunostained with an anti-LC3 antibody (green). Representative images are shown in (D). LC3 puncta are observed in etoposide-treated PPM1D+/+MEFs. (E) The proportion of cells with LC3 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ±  SD (n = 3 experiments). *p < 0.05, **p < 0.01. (F, G) The indicated MEFs were treated with etoposide (10 µM) for 6 hr and then analyzed using electron microscopy. Representative images are shown in (F). Many autophagic vacuoles (arrows) can be seen in etoposide-treated PPM1D+/+cells (upper panel). "N" indicates the nucleus. Bar = 2 µm. A representative autophagosome (AP) and autolysosome (AL) are shown in the insets. (G) The number of autophagosomes and autolysosomes in each cell were counted (n > 8 cells). Red lines indicate the mean value. *p < 0.05. (H, I) Analysis of autophagic flux. The indicated MEFs were treated with etoposide (10 µM) for 6 hr in the presence or absence of bafilomycin A1 (10 nM), and the expression of each protein was examined by western blotting. α-Tubulin was included as a loading control. (I) Semiquantitative analyses of protein expression (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01, NS: not significant.
Figure 2Reduced EC proliferation in the retina (p5) of in TfebiEC-/- mice. Representative images of vessels of the vascular front and vascular plexus in the retina of control and TfebiEC-/- mice stained with anti-iB4 and Ki-67 antibodies (scale bars: 50 µm). Bar graph indicates the percentage of EC Ki-67+ nuclei versus total nuclei co-localized with iB4+ vessels Reduced EC proliferation in the kidney (p17) of in TfebiEC-/- mice. (B) Representative images of vessels of the kidney in control and TfebiEC-/- mice stained with anti-CD31 and Ki-67 antibodies (scale bars: 50 µm). Bar graph indicates the percent of EC Ki-67+ nuclei versus total nuclei co-localized with CD31+ vessels (mice n=6, mean±SEM; **p<0.001 versus control mice by Student's t-test). Podocin staining is shown to glomerular localization. TFEB silencing reduced EC proliferation. Representative graph of scr-shRNA and sh-TFEB ECs treated for 24 hours with FCS (20%) or VEGF-A (30 ng/ml). (C) DNA incorporation of EdU was detected by flow cytometry. The percentage of proliferating cells is indicated (n=4, mean±SEM; ***p<0.0001 versus scr-shRNA ECs by Student's t-test). TFEB silencing reduced EC proliferation. Representative graph of scr-shRNA and sh-TFEB ECs treated for 24 hours with FCS (20%) or VEGF-A (30 ng/ml). DNA content was determined by propidium iodide staining and assessed by fluorescences-activated cell sorter analysis (representative experiment out of 4 with similar results). TFEB silencing reduced human EC morphogenesis. Representative images of tube-like-structure of scr-shRNA and sh-TFEB human ECs stained with phalloidin-555. Bar graph indicates the percentage of phalloidin+ area in sh-TFEB and scr-shRNA ECs (scale bars: 0.25 mm; n=6, mean±SEM; ***p<0.0001 versus scr-shRNA by Student's t-test).
Figure 1A Diagram showing the experimental design. B SDS-PAGE analysis of soluble (Supernatant) and insoluble proteins (Pellet) from human neurons after treatment for one hour with a mixture of RNase A and RNase T1 (A/T1), or vehicle (Ve-). C Protein aggregation (Pellet) after incubation with different ribonucleases or DNase I in the presence of either EDTA or Mg2+. Ribonucleases used were RNase A (A), RNase T1 (T1), a mixture of RNase A and RNase T1 (A/T1), RNase 1f (1f), and RNase V1 (V1).
Pathology of RML-infected mice.Figure 6Pathology of RML-infected miceA-H The pattern of PrPSc deposition (A-D) and GFAP immunostaining (E-H) is presented.I-P A higher power view of H&amp;amp;E stained hippocampus (I-L) and thalamus (M-P) to reveal vacuolation is presented.Data information: Animals of the same genotype are shown within a column, and incubation times of the presented animals with the RML prion isolate are shown (days). Mice expressing PrP S1 and S3 alleles (central columns) exhibit attenuated PrPSc deposition, gliosis and spongy change. Pathological features of the RML isolate are not altered upon sequential passage through TgPrP(S3.88W)-35 mice (see schematic at bottom of the diagram indicating sequential passages and finally, passage back into WT mice, compare the data in first and fourth columns).