EMBO/sd-character-level-ner huggingface dataset:
Rag GTPases control TFEB subcellular localization.Figure 5(A-C) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG along with a control GTPase or the indicated Rag mutants. Cells were deprived of amino acids (top) or deprived and then stimulated (bottom) for the indicated times and stained for FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue). (D) Quantification of the number of cells with nuclear TFEB from each condition in (A-C). (E, F) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG along with Rap2A (E) or the RagsCA mutants (F), subjected to the indicated treatments and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue). (G) Quantification of the number of cells with nuclear TFEB from DMSO‐ and CQ‐treated fields in (E) and (F). In all fields, scale bars represent 10 μm. In all histograms, each value represents mean±s.d. from three independent fields with N=300.
Figure 3Sequence alignment showing the binding site of let-7a miRNA in the inter-stop codon region of AGO1. Effect of let-7a inhibitor on translational readthrough of AGO1. let-7a inhibitor or control inhibitor was transfected in HeLa cells and the expression of Ago1x was determined by Western blot after 48 h. Densitometric analysis of three independent experiments is shown as a bar diagram (mean ± S.E.). *, P = 0.01. Effect of let-7a overexpression on translational readthrough of AGO1. HeLa cells were stably transfected with pri-let-7a overexpression construct. Ago1x expression was determined by Western blot. Densitometric analysis of three independent experiments is shown as a bar diagram (mean ± S.E.). *, P = 0.007. let-7a inhibitor reduces the AGO1 readthrough efficiency. HeLa cells were transfected with let-7a inhibitor or control inhibitor along with AGO1-TGA (or GCA)-ISR-FLuc construct. Readthrough was quantified by measuring FLuc activity, which was normalized to co-transfected renilla luciferase activity. FLuc mRNA expression in HeLa cells treated with miRNA inhibitors is shown (Right). *, P = 0.027. AGO1 ISR with mutated let-7a binding site shows reduced translational readthrough activity. Assay was done as described above in HeLa cells. Mutated sequence is shown (top). ***, P = 0.008. AGO1 ISR with displaced let-7a binding site (AGO1-TGA-disISR-FLuc) shows reduced translational readthrough activity. let-7a binding site was moved 18 nucleotides downstream of the stop codon. Readthrough assay was done as described above in HeLa cells. ***, P < 0.0001.
Figure 6Mechanisms of vascular injury due to NAFLD endothelial chemokine milieu and preferential interaction with effector immune cells via the CXCL12-CXCR4 axis.
Orthoxenograft mouse MPNST models closely resemble primary tumors.Figure 2Orthotopic MPNST xenografts at passages 1 (OT P1) and 4 (OT P4) were histopathologically similar to their corresponding primary MPNST (PT) in hematoxylin-eosin staining of paraffin-embedded tumor sections from patients MPNST-NF1-001 and MPNST-SP-002. Main panels show a general view of the tumors at low magnification (40×); inset pictures were taken at higher magnification (400×). Orthotopic xenograft and primary MPNSTs exhibited similar immunohistochemical features. A representative immunostained section of vimentin, CD34, S100, and Ki-67 is shown for primary tumors (PT) and orthotopic tumors (OT) from patientsMPNST-NF1-001 and MPNST-SP-002. Positive antibody signals are shown in brown, and the hematoxylin counterstain in blue. Main panels show pictures at high magnification (400×); inset pictures show mitotic cells present in these tumors.
Figure 4A. Superposition of the β5i/β6 active sites of genuine mouse iCP and the hβ5i/hβ6 human/yeast chimera illustrates that Met45 adopts the same 'open' conformation in both structures. Amino acid side chains important for substrate binding are depicted as sticks. The catalytic Thr1 is colored in black. Met31, the single amino acid difference between mouse and human β5i active sites, is highlighted in green. B. Superposition of the genuine mouse and the human chimeric β5c/β6 active sites according to panel A proves the proper orientation of Met45 in the 'closed' conformation. C. Superposition of the β5c/β6 and β5i/β6 active sites of the natural mouse cCP and iCP according to panel A. Note the distinct conformations of Met45 (Huber et al, 2012). D. Superposition of the hβ5c/hβ6 and the hβ5i/hβ6 chimeric proteasomes confirm the distinct positions of Met45 analogously to the natural mouse subunits shown in panel C.
Figure 4Functional annotation charts (Benjamini, p<0.05) using GREAT perfomed on the 98 significantly over-expressed genes by CSP-TTK21 treatment in tauopathic mice (TAU MOL vs. TAU VEH). Significance is indicated as -log10(Benjamini p_value) Heatmap representing expression z-score of the 98 significantly over-expressed genes in the TAU MOL vs. TAU VEH comparison, for all experimental conditions. Color-coding was performed according to the z-score of the normalized reads counts divided by gene length. Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson"s dis Venn diagram showing that most of the genes up-regulated by CSP-TTK21 treatment were also down-regulated in the pathology (81/98, p=7.1.10-88, Khi2 test), as well as a significant overlap (p=2.10-29, Khi2 test) genes up-regulated by learning in WT RNA-seq data showing expression of several immediate early genes (IEGs) : Nr4a1, Arc, Egr1, Dusp1, Fos and Junb. Most of the down-regulated IEGs in tauopathic compared to WT mice present a significant induction in THY-Tau22 mice after CSP-TTK21 injection. Adjusted p-values corresponding to p<0.05 are indicated by *, for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH RNA-seq data showing that CSP-TTK21 treatment of tauopathic mice restored the expression of plasticity/memory-relevant target genes : Klotho (Kl), and Neurotensin (Nts). Adjusted p values corresponding to p<0.05 are indicated by *, for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH. F RT-qPCR validations performed in a different cohort of mice from the RNA-seq study (n=4-5/group). One-way ANOVA with uncorrected Fisher"s test. Klotho, Kl: F(3,16)=2.949, p=0.0643; * learning p=0.0145 for WT VEH vs. WT VEH_HC, $ pathology p=0.0395 for WT VEH vs. TAU VEH. Nts: F(3,14)=4.290, p=0.0241; $ pathology p=0.0036 for WT VEH vs. TAU VEH, # molecule p=0.0081 for TAU MOL vs. TA Western blot for Klotho and Neurotensin expression in the different experimental conditions. Immunoblot results are shown (n=5/group). Bar graphs represent the quantification of the protein levels as percentage of the control group WT VEH_HC, arbitrarily set at 100%. Each detected protein was normalized to the corresponding amount of total proteins in the gels or the nitrocellulose membrane. Klotho and Neurotensin were further normalized to the level of actin. One-way ANOVA with uncorrected Fisher"s test. Klotho: F(3,16)=5.192, p=0.0107, * learning p=0.0117 for WT VEH vs. WT VEH_HC, $ pathology p=0.0027 for WT VEH vs. TAU VEH, # molecule p=0.0282 for TAU MOL vs. TAU VEH. Nts: F(3,16)=3.748, p=0.0326, $ pathology p=0.0200 for WT VEH vs. TAU VEH, # molecule p=0.0079 for TAU MOL vs. TA
Figure 7A Schematic representation of cued-fear conditioning experimental procedure. B, C Quantification of activity level and cumulative freezing duration at 12w during seconds 90-210 of day 2 in WT, Syne4-/- mice, Syne4-/- mice injected with AAV.Syne4, and Syne4-/- mice injected with AAV.GFP, n=14 for WT, n=16 for Syne4-/-, n=9 for Syne4-/- + AAV.Syne4, n=5 for Syne4-/- + AAV.GFP. (C) Activity level. (B) Cumulative freezing duration.
Complex Mpl trafficking includes conventional and autophagic secretory pathways, as well as multiple recycling pools.Figure 8A and B) Surface biotinylation and total lysates western blot analysis of HEL or K562Mpl‐mKO2 cells after treatments for 60 min with Tpo, ionomycin (I), PMA, Mon or A23187. C) Total lysate, surface biotinylation and exosomal fraction western blot analysis of HEL and K562Mpl‐mKO2 cells treated with Mon or A23187 for 15 h. D) Western blot analysis of HEL cells treated, or not, with Bafilomycin A1 at 100 nm for 3 h and then stimulated or not with Tpo at 25 ng/mL for 30 min. The quantification of the blot is shown. E) Surface biotinylation and total lysates western blot analysis of HEL cells after treatments for 30 min with Tpo or for 3 h with 3−MA (3−Methyladenine) or Rp. F) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing ATG5 K130R‐V5 6 h post‐transfection. G) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing either GRASP55-V5 or HA-STX5.
Deletion of ERp18 Promotes ER to Golgi Trafficking of ATF6α A-C Wild type cells stably expressing ATF6α (A) or ERp18 KO cells stably expressing ATF6α (B), and ERp18 KO cells stably expressing ATF6α transiently transfected with ERp18 (RV cells) (C), were pre-treated with 30 µM PF429242 (S1P inhibitor) for 1 h prior to induction of ER stress with 10 mM DTT as indicated.Figure 8A-C Wild type cells stably expressing ATF6α (A) or ERp18 KO cells stably expressing ATF6α (B), and ERp18 KO cells stably expressing ATF6α transiently transfected with ERp18 (RV cells) (C), were pre-treated with 30 µM PF429242 (S1P inhibitor) for 1 h prior to induction of ER stress with 10 mM DTT as indicated. D-F Wild type cells transiently expressing ATF6 C467A (D), ATF6 C618A (E), or ATF6 DM (F) were treated as above. Whole cell lysates were separated by SDS-PAGE under reducing conditions and analysed by western blotting for ATF6α. The positions of unprocessed ER-localised ATF6α (ER) and the O-linked glycan modified Golgi-translocated ATF6α (Gol) are indicated as is ATF6-P. G The percentage of Golgi-ATF6α was quantified, the error bars represent ± standard deviation for at least three independent experiments.
Figure 1(A) Schematic overview of pA-DamID. Permeabilized cells are incubated with a primary antibody against a NL component, which in turn is bound by a pA-Dam fusion protein. Nearby DNA is then m6A methylated upon Dam activation by incubation with SAM. The m6A pattern can be visualized by staining with fluorescent m6A-Tracer protein, or mapped genome-wide. Optionally, cells can be flow sorted to isolate specific cell populations prior to genome-wide mapping of m6A. All steps are performed on ice, with the exception of 30-minute Dam activation at 37 °C. (B) Representative confocal microscopy sections of m6A-Tracer signal in HAP-1 cells following pA-DamID with indicated antibodies. pA-Dam only: primary antibody was omitted, the dotted yellow line indicates DAPI segmentation. Free Dam: permeabilized cells were treated with freely diffusing pure Dam protein and SAM for 30 min. Scale bar corresponds to 2 µm. (C) Quantification of peripheral enrichment of antibody staining and m6A-Tracer signals after pA-DamID with indicated antibodies. The nuclear rim and interior were segmented using DAPI signal and the mean m6A or antibody signal was determined and tranformed to a log2-ratio. The nuclear rim mask extends slightly beyond the NL, resulting in underestimation of the real enrichment. (D) HT1080 cells expressing inducible Dam-Lamin B1 were treated with Shield1 to induce m6A methylation. Cells were either fixed immediately or processed according to the pA-DamID protocol (as negative controls) and then fixed on poly-L-lysine coated cover slips. Cells were imaged by confocal microscopy for the NL (Lamin B2 antibody) and methylation (m6A-Tracer) for both conditions. Laser settings were changed between images to optimize image quality. The mask for defining the nuclear rim was obtained by segmentation of the DAPI image.The scale bar corresponds to 2 µm. (E) For every cell, the 50% decay distance from the nuclear periphery was determined for Lamin B2 and m6A-Tracer by fitting exponential decay functions from the nuclear rim as defined by segmentation of the DAPI image The difference in 50% decay distance between Lamin B2 and m6A-Tracer was used as a measure of the thickness of the m6A-Tracer layer. (F) Distribution of the m6A-Tracer layer thickness for HT1080 cells expressing Dam-Lamin B1, visualized before and after the pA-DamID protocol. For comparison, a similar analysis was performed with HT1080 cells not expressing Dam-LaminB1, subjected to Lamin B2 pA-DamID.
The Q623H caPOT1 mutation leads to elongation in hESCs A) Single Telomere Length Analysis (STELA) for POT1 Q623H/Q623H, and POT1 WT/Q623H hESCs compared an unedited wild-type hESC clone that was isolated and cultured in parallel to the edited clones.Figure 2A) Single Telomere Length Analysis (STELA) for POT1 Q623H/Q623H, and POT1 WT/Q623H hESCs compared an unedited wild-type hESC clone that was isolated and cultured in parallel to the edited clones. Numbers above the radiograph indicated days after editing the respective POT1 mutations. B) Quantification of telomere length data from two STELA experiments using the same genomic DNA, shown in (A) and (Appendix Figure 2C). Indicated in red is the mean telomere length and the error bars indicate the standard deviation.
CUL3Δ403–459 displays increased auto-ubiquitylation and ubiquitylation of KLHL3.Figure 2A-D In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation. (A) to determine the relative modification of KLHL3 by CUL3WT and CUL3Δ403-459, KLHL3 was included into the reaction at 2× molar concentration over CUL3WT and CUL3Δ403-459. (B, C) Reactions serve to determine basal auto-ubiquitylation of CUL3WT or CUL3Δ403-459 and do not contain KLHL3 or other potential substrate proteins. Lysine residues on CUL3WT or CUL3Δ403-459 act as the substrate. (B) High molecular weight bands reflect ubiquitin chain linkages on CUL3 or the multiple mono-ubiquitylation of a number of CUL3 lysine residues. (C) Activity assays contain methylated ubiquitin, a form of ubiquitin incapable of forming ubiquitin chains; as such, higher band shifts reflect the attachment of mono-ubiquitin to one more lysine residue on CUL3WT or CUL3Δ403-459, respectively. (D) Activity assay performed as in (C) with methyl-ubiquitin, the boxes shown on the gel are indicative of the gel pieces excised for mass spectrometry analysis in (F). E Schematic representation (to linear residue scale) highlighting the domains of CUL3 and schematic representation of the lysine residues of CUL3WT or CUL3Δ403-459, modified at 5 and 45 min, respectively, in the in vitro ubiquitylation assay shown in (D) and as determined by mass spectrometry. F Structural docking model of CUL3Δ403-459 based on the structure of full-length CUL1 (1LDK) using Chimera (see Materials and Methods). The NTD is coloured green, while the CTD is red, and the four panels sample possible positioning of the CTD relative to the NTD in CUL3Δ403-459, assuming full flexibility of the linker after deletion of three helices encoded by exon 9.
Figure 1(A) Breeding scheme and mouse nomenclature. (B) Number of pups obtained from Nlrp3NeoR-A350V/NeoR-A350V x Fcgr1CreTg/WT matings, with expected and observed Mendelian ratios of the indicated genetic offspring. (C) Survival curves of Nlrp3NeoR-A350V/WT (n=14) and Nlrp3Mac-A350V/WT littermates (n=14). Data information: Statistics were analyzed by (C) log-rank (Mantel-cox) test Error bars represent means ± SD. * p<0.05; ** p<0.01; *** p<0.001. (D) Growth curves of Nlrp3NeoR-A350V/WT (n=8) and Nlrp3Mac-A350V/WT littermates (n=10). Data information: Statistics were analyzed by (D) student t-test Error bars represent means ± SD. * p<0.05; ** p<0.01; *** p<0.001. (E) Representative pictures of Nlrp3NeoR-A350V/WT and Nlrp3Mac-A350V/WT pups at indicated days after birth. Scale bar 1 cm. (F-I) (F) IL-1β, (G) IL-18, (H) IL-6 and (I) TNF analyses in serum from 6- to 9-day old pups (Nlrp3NeoR-A350V/WT n=7, Nlrp3Mac-A350V/WT n=6). Data information: Statistics were analyzed by (F-I) Mann-Whitney test. Dots represent individual biological replicates. Error bars represent means ± SD. * p<0.05; ** p<0.01; *** p<0.001.
The human HOIP K784R mutant as a part of the LUBAC generates linear ubiquitin chains and ubiquitinates its substrate NEMO in vitro and in cells.Figure 2. In vitro ubiquitination assays for the indicated times using the recombinant proteins of ubiquitin (Ub), E1, E2 (UbcH7), HOIP (WT, K784R or C885A mutant), HOIL-1L, SHARPIN and NEMO. Immunoblotting of Linear ubiquitin chains, NEMO, HOIP, HOIL-1L and SHARPIN detected by using antibodies as indicated. Representative data from three independent experiments. B . Immunoblotting to detect ubiquitination of NEMO in HEK293T cells transiently expressing Flag-NEMO, GFP-SHARPIN, and HOIL-1L-HA with Myc-HOIP wildtype (WT), Myc-HOIP K784R or Myc-HOIP C885A. Total cell lysates in denaturing conditions subjected to SDS-PAGE followed up by immunoblotting using antibodies as indicated. Anti-Vinculin antibody to monitor loading. Representative data from three independent experiments.
Aged cac mutant photoreceptor terminals show morphological defects and accumulate late stage AVs.Fig 2A. TEM sections of 3- and 27-days-old photoreceptor terminals of flies. Terminals of 27-days-old flies show terminal expansion, loss of capitate projections (CPs) and active zones (AZs) and increased number of mitochondria per terminal. Scale bars, 500 nm. B. Inset from A representing the photoreceptor terminal boxed in white. Yellow arrow indicates double-membraned autophagosome, red indicates amphisome, and blue indicates two fusion-primed AVs. Scale bars, 500 nm. C-E. Quantification of CPs, AZs, and mitochondria per cartridge, respectively. Data are presented as means ± SEM. F. Quantification of the different AVs per cartridge. The mutant terminals accumulate late stage AVs and fusion-primed AVs (ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001). Data are presented as means ± SEM G. 30-days old fly eye brain complexes stained with neuronal marker Elav (blue) and poly-Ub antibody (red). The right panel is the enlarged images of the boxed regions at the left side H. The level of p62 is increased in cacJ brain. I. Quantification of H. Data are presented as means ± SEM. (*: p < 0.05).
N-terminal domain of Tap-1 is under diversifying evolutionary selection.Figure 6N-termini of Tap-1 share more than 30% overall amino acid sequence identity. The identity of the nucleotide and amino acid sequences across the indicated regions among tap-1 of 36 V. cholerae strains (sliding window = 1) is shown. Green bars indicate 100% identity, yellow bars 30-99%, and red bars < 30%. The percent amino acid identity across the indicated region, with the length of amino acids indicated, is shown at the bottom of the figure. The first 14 amino acids of class 1 and 2 Tap-1proteins were excluded from this analysis. The ratio of percentage nucleotide sequence change to percentage amino acid sequence change over the indicated region is shown in the graph.
Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission.Figure 6.Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. (A-D) Still frames from time-lapse movies of Hep3B cells expressing LAMP1-mCherry. Cells were starved for 2 h in HBSS and subsequently treated for 30 min with either DMSO (A and B) or 40 µM Dynasore (C and D), which induced extensive tubulation of LAMP1-positive compartments. Bars (A-D): 20 µM; (A′-B′) 2 µM; (C′-D′) 10 µM. (E-G) To demonstrate the reversibility of this tubulation, Dynasore-treated cells were washed extensively with drug-free media containing 10% FBS and monitored by time-lapse microscopy for 45 min. Frequently, after drug washout, LAMP1-positive tubules exhibited noticeable varicosities (E and F, arrows; bars, 10 µM) along their length. These sites are suggestive of areas of scission and resumed budding of nascent protolysosomes from the reformation tubules (G; bars, 10 µM). (H) Tubules from cells undergoing drug washout were quantified by tracing their lengths at the beginning and end of these movies. Still frames from a representative movie show tubule content at t = 10 and 45 min after drug washout. Bars, 20 µM. (I) Analysis of five independent movies showed an average decrease in total tubulation of ∼50% after drug washout. Data represent the average relative change in total tubule length between the first and last frames of the time-lapse movies. Error bars represent SE; *, P < 0.05.
Figure 4(A) T47D-MTVL cells transfected with control siRNA or siRNA against BRM and BRG1 (B/B) were induced or not with hormone, and the extracted RNA was hybridized to a gene expression array. Analysis of microarray data found 1359 genes regulated by hormone affected by B/B knockdown (middle panel). In 62% of them (847 genes) B/B is associated with gene repression (right panel). (B) B/B knockdown reduces R5020-induced cell proliferation of T47D-MTVL cells. T47D-MTVL cells were transfected with control or B/B siRNAs and treated with 10 nM R5020 as indicated, and the cell proliferation ELISABrdU Colorimetric assay (Roche) was performed. The percentage of increase in proliferation is shown for each condition. Bars represent mean ± SD (n = 5). * P-value < 0.05; ** P-value < 0.01; *** P-value < 0.001 (C) Based on the analysis of ChIP-seq experiments performed for BRG1 (see materials and methods), the number as well as the distribution of binding regions is shown. (D-E) Genome-wide distribution of BRG1 around all PRBs or PRBs associated to up, down or non-regulated genes (Panels D and E, respectively). The overlapping between PRBs and BRG1 is also shown (Panel D, inset). (F) The overlapping of BRG1 with BAF170 binding regions is shown for T47D cells (left panel). More regions of BRG1 alone were found associated with hormone-repressed genes compared with BRG1-BAF170 and random regions (right panel). * P-value < 0.05.
PrP expression in transgenic mice.Figure 3A Brain homogenates of TgPrP(WT), TgPrP(S1)-17 and TgPrP(S3.F88W)-35 mice were analysed by Western blot before and after PNGaseF digestion using the PrP antibodies Sha31, 12B2 and 9A2. Prnp0/0, and WT brain homogenates were used as controls. B The ratio of C2 to full-length PrP in TgPrP(S3.F88W)-35 and WT mice (left panel, n = 7, P = 1.46E-06) and in RK13 cells expressing WT and S3 PrP (right panel, n = 5, P = 4.01E-05) is shown. Values are presented as mean ± SEM. Unpaired, two-tailed t-test, ****P < 0.0001. C Different tissue homogenates from a TgPrP(S3.F88W)-35 mouse were PNGaseF-digested and analysed for PrP using the antibody 1A6. FL, full-length PrP; ns, non-specific signal detected in the immunoblotting procedure.