EMBO/sd-character-level-ner huggingface dataset:
Figure 3Schematic representation of Rab8a protein. To prevent C-terminal geranylgeranylation, the Cys204 residue was substituted with a serine residue. Alternatively, two truncated proteins (N100 and ∆N100 fragments of Rab8a) were prepared for this experiment. C-terminal geranylgeranylation of Rab8a was dispensable for BAG6 recognition. The N-terminal GTPase domain of Rab8a was essential for BAG6 recognition. A series of Flag-tagged truncated fragments of WT Rab8a were expressed in HeLa cells with S-tagged BAG6 and treated with (+) or without (-) protease inhibitors for 4 h. Flag-Rab8a substrates were immunoprecipitated and probed with an anti-BAG6 antibody. Hydrophobicity recognition domain of BAG6 (N465) was critical for Rab8a recognition. A schematic representation of the BAG6-truncated proteins used in this experiment is shown in the upper panel. Numbers denote the corresponding amino acids of mammalian BAG6. Positions of UBL, BUILD, and DUF3538 domains, which are all linked to hydrophobicity recognition by BAG6 [37], are indicated. WT, wild-type; ∆N465, N-terminal 465 residues-deleted mutant; and Ν465, C-terminal 689 residues-deleted mutant. CHX chase experiments show that GDP-bound Rab8a (T22N) was a highly labile protein, while the GTP-bound active mutant (Q67L) was a stable protein (E). Instability of the T22N mutant was not perturbed by the C204S mutation (F). Actin was used as a loading control. N-terminal 100 residue fragment of the Rab8a GTPase domain was sensitive to the proteasome inhibitor, while the ∆N100 fragment was not. Rab8a (T22N) was polyubiquitinated in the presence of the proteasome inhibitor. Asterisks indicate non-specific bands.
Models of HVGs and LVGs HVGs (top panel) are enriched in environmentally responsive genes, with processes often more variable either during the day (yellow rectangle) or the night (grey rectangle).Figure 6HVGs (top panel) are enriched in environmentally responsive genes, with processes often more variable either during the day (yellow rectangle) or the night (grey rectangle). These genes tend to be smaller, to be targeted by a higher number of TFs (represented by the TFs binding motifs in green) and to have a more compacted chromatin environment (nucleosomes with red rectangles). On the other hand, LVGs (bottom panel) are enriched in genes involved in primary metabolism. These genes tend to be longer, to be targeted by a smaller number of TFs (represented by the TFs binding motifs in green) and to have a more open chromatin environment (nucleosomes with blue triangles), compared to HVGs.
Figure 2A, Representative images of immunofluorescent analyses of USP33 (red) and HIF2α (green) in human primary GBMs. Frozen sections of human GBMs were immunostained with antibodies against USP33 and HIF2α, and counterstained with DAPI to show nuclei (blue). Scale bar, 40 μm. (n = 5 sections; mean ± s.e.m.). The percentage of USP33+ cells in the HIF2α+ population was quantified. The majority of HIF2α+ cells were positively stained for USP33 in human GBMs. C Immunoblot analysis of HIF2α induction in T4121 GSCs expressing shUSP33 or shNT in response to low oxygen. Twenty-four hours post lentiviral transduction of shUSP33 or shNT, GSCs were incubated under 20% or 5% oxygen for 24 hours and then harvested for the immunoblot analysis. Disruption of USP33 abrogated HIF2α induction in GSCs in response to 5% oxygen. D Immunoblot analysis of HIF2α induction in T4121 GSCs expressing shNT or shUSP33 in response to CoCl2 mimicking hypoxia. Twenty-four hours post lentiviral transduction of shUSP33 or shNT, GSCs were treated with CoCl2 (300 µM) for 12 hours and then harvested for the immunoblot analysis. Disruption of USP33 abrogated CoCl2-induced HIF2α expression in GSCs. E Immunoblot analysis of HIF2α protein levels in GSCs expressing ectopic Flag-tagged USP33 in normoxia. Cells were transduced with lentiviruses carrying Flag-tagged USP33 or the control vector, followed by two rounds of puromycin selection. Ectopic expression of USP33 elevated HIF2α protein levels in GSCs cultured under 20% oxygen. F Immunoblot analysis of ectopic HIF2α protein levels in 293T cells with gradually increased amount of ectopic USP33. 0.5 µg of HA-tagged HIF2α plasmids together with the increased amounts of GFP-tagged USP33 plasmids (0, 0.1, 0.2 or 0.3 μg) were introduced into 293T cells. Forty-eight hours post transfection, levels of ectopic HIF2α-HA and GFP-USP33 were analyzed by immunoblot analysis. Concomitantly increased levels of ectopic HIF2α and USP33 expression indicated the positive regulation of HIF2α protein by USP33. G Immunoblot analysis of ectopic HIF2α protein levels in 293T cells expressing ectopic wild type (WT) USP33 or the catalytically dead (CD) USP33. HA-tagged HIF2α plasmids (0.5 µg) were introduced into 293T cells alone or together with GFP-tagged WT or CD USP33 plasmids (0.2 µg). Levels of ectopic HIF2α-HA and GFP-USP33 were analyzed 48 hours post transfection. Expression of the wild type USP33 but not the catalytically dead USP33 increased the ectopic HIF2α protein levels.
Atg8F77/79A does not undergo cleavage in the carboxyl terminus.Figure 3(A) Atg8WT-HA, Atg8F77A-HA, Atg8F79A-HA or Atg8F77/79A-HA was transformed into Δatg8 cells. Cell extracts were prepared and subjected to 13.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) containing 6 M urea, followed by immunoblot analysis with anti-Atg8 antibodies. (B) Prediction of protein-protein interaction sites in Atg8 ( Neuvirth et al, 2004). A three‐dimensional model of Atg8 is coloured according to ProMate prediction ( see supplementary information online) with red representing the predicted protein-protein binding sites, and Phe 77 and Phe 79 are marked in green within the prediction site. (C) Pull‐down assay of Atg8 mutants with Atg4. His-Atg4 was attached to cobalt beads (Talon metal‐affinity resin), washed and incubated with total cell extracts of Atg8 mutants. Elution was subjected to SDS-PAGE followed by western blot analysis using anti‐Atg8 and anti‐Atg4 antibodies. (D) Localization of green fluorescent protein (GFP)-Atg8wt and GFP-Atg877/79 cells carrying Atg4 plasmid under normal (control) and starvation conditions. Arrows point to the pre‐autophagosomal structure, and arrowheads point to GFP within the vacuoles. (E) Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐API antibodies. mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.
Figure 11During normal conditions, the translation of CHOP protein is blocked by huORFchop-TI due to ribosomes stalled at 81-90 nts within the 105-nt huORFchop transcript. However, Endouc/ENDOU is quickly produced when cells encounter environmental stress, e.g., heat-shock and hypoxia as in the present work. They bind to the hairpin structure of huORFchop transcript formed at 72-91 nts and cleave it at the 80G-81U site. This endonuclease activity changes the conformation of inhibitory structure formed by huORFchop transcript in turn releasing the stalled ribosomes to scan through the huORFchop transcript and re-initiate at AUG of the main downstream coding sequence (DCS) of huORFchop transcript. Finally, CHOP transcripts were continuously translated via the IRES activity driven by huORFchop -69-105-nt and the increase of p-eIF2α induced by Endouc/ENDOU.
Induction of autophagy in macrophages restricts legA9 mutant replication, and rescues colocalization with LC3 and fusion with lysosomes.Figure 3Induction of autophagy in macrophages restricts legA9 mutant replication, and rescues colocalization with LC3 and fusion with lysosomes. BMDMs were untreated or pretreated with rapamycin (Rap) for 1 h and kept throughout the infection. BMDMs were infected with L. pneumophila JR32 or the leg A9 mutant for (A) 1 or (B) 24 h with an MOI of 0.5. CFUs were scored at these time points. (C, D) Percent colocalization of L. pneumophila with (C) lyso‐tracker and (D) LC3. Hundred bacteria were scored from each coverslips. (A-D) Data are presented as mean + SD of n = 3 and are representative of three independent experiments. Asterisks indicate significant differences (**p < 0.01; two‐tailed t‐test).
ORF57 contributes to the upregulation of circHIPK3 levels(A) qPCR analysis of circHIPK3 levels at time points in TREx post-induction with GAPDH as a housekeeper (n=3).Figure 4(A) qPCR analysis of circHIPK3 levels at time points in TREx post-induction with GAPDH as a housekeeper (n=3). (B) qPCR analysis of HIPK3 levels at time points in TREx post-induction with GAPDH as a housekeeper (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (C) qPCR analysis of circHIPK3 expression in HEK-293Ts transfected with GFP, ORF50-GFP, ORF57-GFP or ORF-57-GFP RGG1/2 with GAPDH as a housekeeper (n=3). (D) qPCR analysis of HIPK3 expression in HEK-293Ts transfected with GFP, ORF50-GFP, ORF57-GFP or ORF-57-GFP RGG1/2 with GAPDH as a housekeeper (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (E) qPCR analysis of circHIPK3 expression in HEK-293Ts transfected with 4 µg GFP, or 0.25-4 µg ORF57-GFP, GAPDH was used as a housekeeper (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (F) qPCR analysis GFP RIPs in GFP or ORF57-GFP transfected HEK-293Ts at 16 and 24 hours post-transfection respectively, n=3 for each time course. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.
Figure 4Volcano plots displaying the genes that are differentially expressed in immature enterocytes 2 upon SARS-CoV-2 infection of colon organoids. infected vs. mock infected cells The statistical significance (-log10 adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in blue in all panels are gene names of selected differentially expressed genes between the compared two populations. Labeled dots in red in all panels are gene corresponding to interferon if detected. Volcano plots displaying the genes that are differentially expressed in immature enterocytes 2 upon SARS-CoV-2 infection of colon organoids. bystander vs. mock infected cells The statistical significance (-log10 adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in blue in all panels are gene names of selected differentially expressed genes between the compared two populations. Labeled dots in red in all panels are gene corresponding to interferon if detected. Volcano plots displaying the genes that are differentially expressed in immature enterocytes 2 upon SARS-CoV-2 infection of colon organoids. infected vs. bystander cells. The statistical significance (-log10 adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in blue in all panels are gene names of selected differentially expressed genes between the compared two populations. Labeled dots in red in all panels are gene corresponding to interferon if detected. D. Dot plot of the top 42 most differentially expressed genes upon SARS-CoV-2 infection in mock, infected and bystander cells at 12 and 24 hpi. The dot size represents the percentage of cells expressing the gene; the color represents the average relative expression across the cell type. E. Same as in (D) but for the top 30 most differentially expressed ISGs. F-J. Same as (A-E) but for ileum organoids. Labeled dots in blue in all panels are gene names of selected differentially expressed genes between the compared two populations. Labeled dots in red in all panels are gene corresponding to interferon if detected.
Figure 1A. Confocal microscopic image showing HeLa cells immunostained for Nup358 (green, upper panel) or Nup214 (green, lower panel) and RanGAP1 (red) using specific antibodies. DNA was stained with Hoechst 33342 (blue). Scale bars, 10 μm. B. COS-7 cells transfected with GFP-Nup358 and one of the cells was subjected for live imaging using confocal microscopy and the frames at indicated time points have been provided. Arrows denoted by different colors indicate individual AL structures. Note that one of the AL structures (marked by yellow arrow) originated from the NE (dotted line) and fused with another AL structure (marked by red arrow), which later fused with a different AL structure (marked by blue arrow). N, nucleus; C, cytoplasm. Scale bar, 5 μm. C. Maximum intensity projection confocal image of a sodium arsenite treated HeLa cell immunostained for endogenous Nup358 (green), P bodies (red, Dcp1a as a marker) and SGs (blue, eIF3η as a marker) using specific antibodies. Scale bar, 10 μm. The histograms represent fluorescence intensity profile along the dotted arrows. Adjacent graph represents quantitative data showing percentage of P bodies (top) or SGs (bottom) associated with nuclear envelope (NE), Nup358 positive AL, with each other or unassociated with any of the other mentioned structures (others). Data are presented as mean SD (n = 3). D. HeLa cells were transfected with Nup358 siRNA (siNup358) or Nup214 siRNA (siNup214). Cells were fixed and stained for endogenous Nup358 (green, upper panel) or Nup214 (green, lower panel) and endogenous P body marker (Dcp1a, red). Graph represents the quantitative data as mean ± SD. The data was obtained from three independent experiments, and in each experiment, 100 cells were counted from different fields for the presence of intact microscopically distinct P bodies and expressed as percentage. D. HeLa cells were transfected with Nup358 siRNA (siNup358) or Nup214 siRNA (siNup214). Western blot (WB) indicates the extent of Nup358 depletion and the level of Dcp1a.
a) Effects of expression of wild-type (WT) CerS1 and its catalytically inactive V5-tagged mutant (Mut), which cannot generate C18-ceramide, on the formation of LC3B-II were determined by western blotting (+/− tet).figf3(a) Effects of expression of wild-type (WT) CerS1 and its catalytically inactive V5-tagged mutant (Mut), which cannot generate C18-ceramide, on the formation of LC3B-II were determined by western blotting (+/− tet). Successful induction of wild-type and mutant CerS1 expression was confirmed using anti-V5. β-actin was used as a loading control. Full blots can be found in Supplementary Figure 13. (b) Generation of endogenous C18- and C18:1-ceramides in response to wild-type CerS1 and mutant CerS1 induction was measured by LC/MS/MS. (c) Effects of wild-type CerS1 and C18-ceramide induction (+ tet) on GFP and LC3B-GFP lipidation were visualized using confocal microscopy and compared to effects of noninduced controls (− tet). Scale bars, 10 μm (d) The effects of induction of wild-type CerS1 and C18-ceramide and catalytically inactive mutant CerS1 (+ tet) in the regulation of mitochondrial function were assessed by measuring OCR using the SeaHorse and compared to roles of noninduced controls (− tet). (e) Targeting mitochondria with autophagolysosomes in the absence (− tet) or presence (+ tet) of wild-type CerS1 and C18-ceramide was visualized by colocalization of MTG and LTR using confocal microscopy. Scale bars, 10 μm. (f) Effects of wild-type and mutant CerS1 (−/+ tet) on ATP generation. (g) Effects of wild-type CerS1 (−/+ tet) on ATP generation in the absence (control shRNA) or presence of shRNA-mediated knockdown of LC3B. Data shown are an average of at least three experiments ± s.d. (*P 0.05).
Figure 2Three constructs were designed to target GmLMM1 and its close homologous genes. The CRISPR target sequence is shown in orange, and the corresponding protospacer adjacent motif (PAM) site is shown in blue. The box indicates the putative sites shear cut by cas9. Two representative T1 transgenic lines (aged 1 month) are shown for each construct. The editing results were examined by PCR and sequencing; - means deletion of the corresponding nucleotide. The red or green face-labeled letters indicate the mutated or added nucleotides, respectively. (A) Editing of GmLMM1 and Gm13G053800 by construct 1 (C1). The alignment of the sequence and phenotypes of two T1 lines (C1-16-1 and C1-16-5) generated from the same T0 line (C1-16) are shown. (B) Simultaneous edits of GmLMM1, Gm13G053600, and Gm13G053800 by the C2 vector. (C) Editing of GmLMM1 and Gm13G053600 by C9 vector.
Figure 4A-H Intracellular localization of LAMP1-positive EL, total (HA) and polymeric (2C1) ATZ in WT MEF exposed to BafA1 for 12 h (A); in Atg4BKO MEF (B); in Atg7KO MEF (C); in Fip200KO MEF (D); in Ulk1/2 double-KO MEF (E); in Atg13KO MEF (F); in Atg9KO MEF (G); in RubiconKO MEF (H). Scale bars: 10 μm. I Quantifications of A-H (n=10, 9, 9, 10, 7, 15, 9, 12 cells). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001.
Figure 5A, CheY crosslinks with FliM∆N-YPet in vivo. The cytoplasm of fliM∆N-YPet ΔcheZ cells overexpressing CheY (strain EW694) or FliMN-CheY (strain EW697) from a plasmid was crosslinked by glutaraldehyde and resolved by SDS-PAGE. 1, 2, 3 stand for three different experiments that underwent this procedure. The plus sign before ~14 and ~15 stands for +55 kDa of FliM∆N-YPet. Note that gel running suffered from parabolic distortion in band positions. The annotations relate to the lowest positions of the bands. The gel was imaged for FliM∆N-YPet fluorescence. To see the crosslinking products, the intense fluorescence of monomeric FliM∆N-YPet is shown at saturation. B, Predicted binding interfaces of CheY (gray ribbon) and FliMM (green spheres) in E. coli. R94 of FliM is shown in blue. Part of the hydrophobic region of FliM (including E214) is shown in orange. C, Clockwise rotation of tethered fliM(R94S)∆N ∆cheZ cells and fliM(R94L)∆N ∆cheZ cells expressing FliMN-CheY (strains EW731 and EW733, respectively) in the absence or presence of acetate (10 mM, pH 7.0). FliMN-CheY concentration was estimated to be ~4 mM by extrapolation of the calibration curves for which a similar CheY expression system was used. No clockwise rotation was observed when benzoate substituted for acetate. Each data point is the mean of a separate experiment. Black line is the mean of all experiments. D, Clockwise rotation of tethered fliM(E214W)∆N ΔcheZ cells (strain EW718) at various concentrations of FliMN-CheY. The concentrations shown are estimates based on the calibration curve , for which a similar CheY expression system was used. Each data point is the average of all measurements at similar FliMN-CheY concentrations, weighted by the sample number of each experiment. E, Distribution of clockwise intervals of fliM(R94SL)∆N ∆cheZ cells expressing FliMN-CheY (strains EW732 and EW734) in the presence of acetate (10 mM, pH 7). F, Distribution of clockwise intervals of fliM(E214W)∆N ∆cheZ cells expressing FliMN-CheY (strain EW718) in the absence of acetate.
a, ChIP-qPCR analysis of REST binding to target genes in isolated PFC neuronal nuclei.f2a, ChIP-qPCR analysis of REST binding to target genes in isolated PFCneuronalnuclei. Shown are young adult (Young), normal aged (Aged) and AD cases, which were stratified as AD1 (MMSE score > 18) and AD2 (MMSE score 8). Also shown are control ChIP assays with non-specific IgG, and a RESTChIP with PCR primers directed 10 kb 3′ to the SNAP25 RE1 site (SNAP25-10K down). Values represent fold change relative to the mean young adult value, and represent the mean ± s.e.m., n = 7. *P 0.05 by the Mann-Whitney test. b, mRNA expression of REST target genes determined by qRT-PCR of PFC. Shown are REST target genes related to cell death pathways and AD pathology (left), and neurotransmission (right). Values are normalized to the mean young adult expression level (100%) and represent the mean ± s.e.m. Young, n = 9; Aged, n = 10; AD1, n = 10; AD2, n = 10. Asterisks above the 'Aged' bars indicate significance relative to young adults; brackets indicate significance of Aged versus AD1 or AD2. *P  0.05 by the Mann-Whitney test. c, Linear regression analysis of nuclear REST versus levels of protein targets or histone H3K9ac in double-labelled PFC neurons. d, Representative images showing inverse relationships between nuclear REST and H3K9ac, PS2, DAXX and BAX in AD1 neurons. Scale bar, 20 µm. e, Global epigenetic regulation in ageing and AD. Histone modification H3K9ac was determined in isolated PFCneuronalnuclei. Values are normalized to the mean of the young adult group (100%), and represent the mean ± s.e.m., **P 0.005 by Student's unpaired t-test. Young, n = 8; Aged, n = 11; AD1, n = 4; AD2, n = 4.
Figure 3P1 - P6 refer to individual patients. Representative examples of the quadruple immunofluorescence quantification of skeletal muscle fibres before and after treatment. The box in the top right-hand corner indicates the normal range, using two antibodies to NDUFB8 and COX I. Quantitative quadruple immunofluorescence of skeletal muscle fibres in the six patients showing a decrease (two-sided Wilcoxon signed-rank test with an empirical level of significance) in the proportion of complex IV deficient skeletal muscle fibres before and after 12 weeks of treatment (empirical P-value = 0.048). Hierarchical cluster analysis of skeletal muscle RNA-seq transcripts before and after treatment showing no clear separation between untreated and treated patients. Volcano plot depicting differentially expressed RNA-seq genes at 10% FDR after 12 weeks of treatment. Significantly different mammalian mitochondrial genes (MitoCarta 2.0) are annotated. Reactome Pathway Analysis of the differentially expressed genes at 10% FDR after 12 weeks of treatment (see Expanded View document for details). Percentage level m.3243A>G in blood, urinary sediment and skeletal muscle. No differences were detected by using the two-sided Wilcoxon signed-rank test with an empirical level of significance. W = week of the study. Cardiac parameters before and after treatment (two-sided Wilcoxon signed-rank test with an empirical level of significance). ES = end-systolic volume (ml) (empirical P-value = 0.047) and index (ml/m2) (empirical P-value = 0.046), measured by MRI. Horizontal lines on (B), (F) and (G) refer to statistical comparisons of the mean values, where ns = non-significant, *P-value ≤ 0.05. For normal reference ranges see (Hollingsworth et al., 2012)
Figure 6(A) Venn diagrams showing overlap of proteins identified in EVs isolated from CSF of control (blue) and LPS-injected (yellow) mice that were present in the two independent replicates. (B) Venn diagrams showing overlap of the CSF EV proteome (control and LPS; purple) with the Mus musculus proteome list available on the EVpedia website (green). (C) GO enrichment analysis using DAVID of the EV proteome compared with total choroid plexus proteome. (D) GO enrichment analysis using DAVID of the EV proteome following LPS treatment compared with total choroid plexus proteome. The grey bars represent the number of proteins and the black dots the p-values. (E-F) DAVID (E) and Ingenuity pathway analysis (IPA) (F) on the CSF-derived EV proteome, taking into account all proteins that are exclusively detected after LPS stimulation in both replicates. The grey bars represent the number of proteins and the black dots the p-values.