EMBO/sd-character-level-ner huggingface dataset:
(G) LN229 cells stably transduced with 6x-Histidine-tagged ubiquitin (His-Ubi) were treated with DMSO or thapsigargin (TG). These cells were lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. Both arrowhead and arrow point to the mono-ubiquitinated Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.
G. Kymographs showing the movement of YFP-EML4-ALK V1 WT, YFP-EML4-ALK V3 WT and YFP-EML4-ALK V3 KD over the duration of the observation period. Scale bars; 1 μm horizontal; seconds vertical.
Immunoblot analysis of 2 pairs of colon tumors from mice treated with or without TAM.
(H) Graph represents Spindly levels at unattached KTs for cells Spindly levels were determined relative to CID and all values were normalized to the mean fluorescence intensity quantified in PoloWT-EGFP expressing cells, which was set to 100% (n≥1282 KTs from at least 59 cells for each condition, n=2 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.
Internalized levels of BACE1 after 20 minutes of HA antibody uptake in HA-BACE1-mCherry transfected WT and KO neurons, co-expressing either eGFP-RAB4 or eGFP-RAB4-S22N pWTRAB4 vs pWTRAB4S22N=0.450, 27-37 neurons per condition, N=3 biological replicates). Scale bar: 5 µm. Data information: Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square.
THP-1-derived ANG translocating into HCT116KO through PLXNB2 in co-cultured system as showed by western blotting. Neamine, a blocker of ANG and PLXNB2 interaction; siCtrl: control siRNA; siPLXNB2-1 and siPLXNB2-2
(B) Venn diagram showing overlap of upregulated genes at individual time-points.
N, Gene set enrichment plot analysed from PCa patients of the PRAD TCGA dataset using the UPR gene signature
Ki67 and cleaved caspase3 staining of representative colons obtained from mice on day10 of colitis induction as described in Figure 2A. The number of proliferating cells per crypt in (A) (upper panel) or apoptotic cells per field in (B) was determined (n=5 per group, 5 crypts were counted per mouse). Scale bar, 50μm.
Rescue of the TPC-KO dynamin defect by the dynamin activator, Ryngo-1-23 (80 μM): mean number of internalised beads per cell as a percentage of the WT control; n = 128-241 cells, **P<0.01, ***P<0.001.
The model suggests the most likely interactions of CheY at the switch. Yellow ring, FliN. Green or red ring, FliMM in a conformation to which CheY can or cannot bind, respectively. Red background region is counterclockwise rotation. Yellow background region is frequent switching. Green shaded region is clockwise rotation. A, When the motor rotates counterclockwise, CheY most likely binds to FliMN because FliMN is a high-affinity site and because it is the most accessible site. Even though FliN is accessible to CheY, an interaction of CheY with FliN is not probable at physiological CheY concentrations, unless CheY is first trapped and potentiated by binding to FliMN. (Potentiated CheY is marked with an asterisk.) FliMM is probably sterically blocked for CheY binding or is in a conformation that does not favor CheY binding. B, FliMN-bound CheY has high probability of interacting with FliN. This interaction generates brief episodes of clockwise rotation, because the complex CheY-FliN is unstable and is likely to quickly dissociate. C, During the short episodes of clockwise rotation shown in B, FliMM either becomes accessible to CheY or is changed to a conformation that favors CheY binding. As a result, CheY (or, most likely, CheY~Ac) binds to FliMM. This binding stabilizes the clockwise conformation of FliMM and prevents the latter from resuming its counterclockwise conformation or becoming again sterically blocked for CheY binding. As long as CheY is bound to FliMM, CheY dissociation from FliN no longer affects clockwise rotation. CheY dissociation from FliMM when CheY is already dissociated from FliN would cause an immediate return to counterclockwise rotation.
A-C Micrographs of sections of E15 cerebral cortex 2 days after IUE with plasmids and magnifications indicated. V= ventricle; VZ: ventricular zone. Scale bars: 20 µm and 10 µm for magnifications. Yellow dotted shapes show examples of heterochromatin spots in nuclei of electroporated cells from the VZ and the non-VZ.
(D) Bacterially produced full-length β-catenin WT and alanine ("A") substitution mutants were purified and reacted with Flag-Plk1 WT produced from mammalian cells in the presence of [γ-32P]ATP. Incorporation of 32P into each "A" mutant or full-length WT β-catenin was confirmed by SDS-PAGE followed by autoradiography analysis.
(B) Left, structure of Paired RAG STC with RAG2 omitted (top view). RAG1 protein is shown as an electrostatic surface and target DNA in cartoon mode with the backbone colored yellow and base colored cyan. The white arrow indicates the positively charged surface for target DNA binding on RAG1. Right, structure of the Paired RAG STC with one RAG2 shown (45° rotation relative to (A)). RAG1 is shown as a molecular surface and colored white. RAG2 is shown as an electrostatic surface. The white arrow indicates the positively charged surface for target DNA binding on RAG2.
F. Relative levels of metabolites in glutamine metabolism. n=9 biological repeats; upper whiskers, maximum-the third quartile; lower whiskers, the first quartile-minimum; boxes, the third quartile-the first quartile; the central line indicates the median; p-values were calculated using two-sample t-test.
Serial dilutions of the strains indicated were spotted on SD C+/- and incubated at 30 oC for 6 d.
(c The HDX of inactive BakΔTM subtracted from the HDX of BidBH3-activated BakΔTM, both bound to nanodiscs, is color coded on the BakΔTM structure
D) Human VPS34 complex II homology model showing VPS15 (green), VPS34 (grey), Beclin1 (beige) and UVRAG (pink) and potential ULK substrates (red spheres). (Left) Y-shaped VPS34 complex II in standard orientation with N and C termini of VPS15 labelled. The blue dashed line represents a lipid membrane, positioned close to the membrane binding (left- and right-hand tips of Y) and lipid kinase (right hand tip of Y) domains of the complex. (Right) Rotated 90 degrees on horizontal axis towards point of view.
Cells were infected for 15 h, then labeled using antibodies to NDP52 and nsP2 (B), or to TGN46, nsP2 and nsP3 (C).
B-D. TRAPP stimulated guanine nucleotide exchange on purified Rab18, Rab1 and Rab2 GST fusion proteins measured by release of 3H-GDP that was pre-loaded onto the Rab proteins.
HepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control (Ctrl). n=3. Results represent three independent experiments. (B) Protein levels of palmitoylated CD36. Data information: ** P < 0.01. Results are shown as mean ± SD. Student's t test (B
Scheme of the encapsulation setup and illustration, on the right, of the distribution of species within the encapsulated LLPS system.
Schematic diagram of orthotopic re-transplantation of cell lines into the lung of recipient wild type C57BL/6J mice.
(b) Phosphorylation of Beclin1 by ROCK1. Recombinant ROCK1 was used for in vitro kinase assay in the presence or absence of Y27632, using recombinant His-Beclin1 (left) or Flag-Beclin1 immunopurified from transfected HeLa cells (right). Proteins were resolved by SDS-PAGE; phosphorylated protein was visualized with autoradiography, and Beclin1 by Coomassie staining.
D HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, then infected with Shigella-mCherry for 2 h for live confocal microscopy. Time frame sequence shows a septin cage assembling around Shigella. Mitochondria (circle) support sites of septin ring assembly around the bacterium. Each frame was acquired every 2 min. The scale bar represents 1 μm. See also Movie EV1.
(B) Genome-wide binding of Pax5-Jak2 in in vitro cultured pro-B cells from Pax5Jak2/+ Rosa26BirA/+ mice at the age of 3 weeks (expressing Pax5) and ex vivo Pax5Jak2/+ Rosa26BirA/+ B-ALL tumors (lacking Pax5), as determined by Bio-ChIP-seq analysis The DNA-binding pattern of full-length Pax5 was determined by Bio-ChIP-seq analysis of ex vivo sorted Pax5Bio/Bio pro-B cells, which carried a C‐terminal biotin acceptor sequence together with an IRES‐BirA gene insertion in the 3' untranslated region of Pax5 Two independent Bio-ChIP-seq experiments were performed for each cell type. Representative binding patterns of Pax5 and Pax5-Jak2 in the three B cell types are shown for a selected genomic region, with horizontal bars indicating Pax5 or Pax5-Jak2 peaks that were identified by MACS peak calling (left). The number of Pax5 (white) and Pax5-Jak2 (grey or black) peaks, which were defined by stringent MACS peak calling with a P value of < 10-10 in the three B cell types, are shown to the right.
(F) HeLa cells were transfected with pmStrawberry-ATG16L1 and ATG9L1-pEGFP, and 5 min movies were recorded. Representative images from the movies are shown.
(C) Gene expression analysis of Stat3+/+ cells, Stat3-/- cells and two Stat3 rescue clones (Stat3.A/B) cultured in absence or presence of LIF. Note that both clones respond to LIF and activate Stat3 direct target Socs3.
(b) Effect of wild-type and mutant UVRAG on Beclin1-mediated autophagy. MCF7.vector, MCF7.Beclin1 and MCF7.Beclin1ΔECD cells were cotransfected with wild-type UVRAG or its mutant expression vector together with GFP-LC3 expression vector. At 16-18 h posttransfection, cells were incubated under normal or starvation conditions for 4 h. Autophagy was quantified as described in Fig. 3. The scale bars represent 20 μm.
h) Example of co-staining for PV (green), miR-29a (red) and nuclear marker (blue) in a P120 mouse. White triangles represent PV+/miR-29a+ cells. Scale bar 20 µm.
D. HeLa cells were initially transfected with control (siControl), Dicer (siDicer) or Nup358 (siNup358) specific siRNAs, followed by FL constructs containing wild type (HMGA2-wt) or mutant (HMGA2-mut) HMGA2 3'-UTR, along with RL as internal control. Graph was plotted using data from three independent experiments. FL/RL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.
(E, F) Co-immunoprecipitation of ectopically expressed Flag-p65 1-310 fragment (containing RHD domain) (E) rather than Flag-p65 311-551 fragment (lacking RHD domain) (F) with endogenous CDC20 in HEK293T cells.
Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate.
Western blotting shows that Flag-Piwi (F-Piwi) WT (WT) and PAZ mutant (PAZmt) but not MID mutant (MIDmt) bound with Armi in Zuc/Piwi-depleted OSCs (Zuc KD + Piwi KD).
Immunoblot analysis of the distribution of the indicated proteins in the CD4+ T cell fraction.
(A) Representative images from HC assay of HeLaTFEB-GFP cells transfected with control (siCTRL) or siPPP3CB, and subjected to the indicated conditions. Analysis of TFEB nuclear translocation in HeLaTFEB-GFP cells transfected with siCTRL or siPPP3CB and treated with DMSO or NSC668394, silenced for EZRIN (siEZR) or serum-starved (stv). Both pharmacological inhibition and silencing of Ezrin induced TFEB nuclear localization in stable HeLaTFEB-GFP cells. Starvation (stv) was used as control. Nuclear translocation of TFEB in stable HeLaTFEB-GFP cells subjected to the indicated conditions is rescue after silencing of PPP3CB. Scale bar 5 μm.
C,D. Quantification of glucose (C) and lactate (D) concentration from media. The media was collected after incubated with mature brown adipocytes for 48 h. n=3 independent experiments in (C), n=6 independent experiments in (D). Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.
D: Addition of the proteasome inhibitor (bortezomib) rescues the loss of protein expression following 8 or 18-hour mithramycin treatment. BT12 cells were treated for 8 or 18-hours with solvent (S), mithramycin (M, 100nM), bortezomib (PI, 2.5μM), or combination of 100nM mithramycin and 2.5μM bortezomib (C).
BMDMs from WT and CYLD KO mice were pretreated with Caspase-1 inhibitor, Z-YVAD-FMK, for 30 min, primed with LPS, and then stimulated with Nigericin for 30 min. ASC specks were assessed by Immunofluorescence with antibody against ASC and the percentage of ASC speck-positive cells was quantified.
(F) Correlation between H3K27me3 and H4K20me1 accumulation after 24h of DOX treatment at initially active gene bodies (left), inactive gene bodies (middle) and 10-kb intergenic windows spanning the X chromosome (right). All scales are logarithmic. All correlations (ρ) are with p<0.01 from Pearson's correlation test.
Representative images of liver sections stained with α-SMA from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.
(a) Representative confocal micrographs of mitochondrial morphology in MEFs of the indicated genotype 24 h after transfection with mtYFP. Where indicated, cells were starved for 2.5 h. Scale bar, 20 μm. (b) Morphometric analysis of mitochondrial shape. Experiments were carried out as in a. Data represent mean ± s.e.m. of three independent experiments (n=100 cells per condition in each experiment).
B) The indicated YFP-FoxO3 constructs were transfected into HeLa cells, purified using YFP resin and PP2A-B56 binding determined by Western blotting. PP2A-A; scaffold subunit.
(I and J) Autophagic flux determination by vacuolar protease-dependent GFP liberation after ACS2 overexpression compared to empty vector controls (Control) similar to (G) and (H). Representative immunoblots at indicated time points (G) and quantification by densitometry of free-GFP/GFP-Atg8p signal ratios (H) are shown. Data represent means ± SEM (n = 4).
g, A small but significant quenching reaction was detected (with 10 mM Fe2+) in R403C-TRPML1Va-expressing cells.
Tracing of [U-13C] L-glutamine ([U-13C] Gln) into indicated metabolites. NIH-3T3 cells were treated with TGFβ (2 ng/mL for all experiments) or mock for 48h, and the medium was replaced (including all treatments) with DMEM lacking L-glutamine and supplemented with [U-13C] Gln for the last 8h. Metabolic steady state was reached at this time point (see kinetic labeling curves in Figure EV3b). Metabolites were measured by LC-MS. The left graph shows the total pool size normalized to the packed cell volume; the right side shows the percent labeling. αKG, alpha-ketoglutarate; AU, arbitrary units.
g, Fold replication of S. Typhimurium in HeLa cells expressing the indicated galectin 3 variants and transfected with the indicated siRNAs. At 2 h and 6 h after infection, cells were lysed and bacteria counted on the basis of their ability to form colonies on agar plates. Galectin 3 proteins are further characterized in Supplementary Fig. 4b. Mean and s.d. of duplicate coverslips>100 bacteria counted per coverslip. Data are representative of at least two repeats. *P< 0.05, Student's t-test. Scale bar, 10 µm.
C. SDS-PAGE showing a representative fraction of the purified spMis18 N-terminal Yippee-like globular domain (amino acid residues 1-120; spMis181-120).
(b) GFP-LC3-expressing RPE cells were depleted of myosin VI by siRNA. Cells were pulse labelled with Texas-red-dextran (red) for 4 h, before chase into fresh medium containing 1 μM MG132 for 2 h. Cells were processed for immunofluorescence microscopy and immunostained for GFP (green). Nuclei were labelled with Hoechst (blue). The insets represent higher magnifications of the areas outlined in the main panels. (c) Mander's overlap coefficients for the degree of LC3 signal co-localizing with dextran were calculated using Volocity software. The results represent >20 cells from n = 3 independent experiments and are illustrated as a box and whisker plot. The box represents the median, 25th and 75th percentiles and whiskers represent the maximum and minimum.
CPEcells form a tight barrier between the peripheral blood and the brain. CPEcells are highly secretory and secrete exosomes from MVBs into the CSF. Systemic inflammation induces activation of the exosome machinery, resulting in an increased released of miRNA-containing exosomes into the CSF. Next, the exosomes cross the leaky ependymal cell layer that line the ventricle and are taken up by astrocytes and microglia, whereupon they transfer a pro-inflammatory message.
E) Representative sanger sequencing chromatogram of four hCD34+ cell-derived heterozygous POT1-edited colonies shown in (C) and (D). WT: wild-type, MT: mutant. The red line indicates the CAS9 cleavage site, 3nt upstream of the PAM site.
A Ccq1 recruits CLRC and SHREC to subtelomeric chromatin, which counteracts nucleosome instability conferred by the DNA sequence of TAS. CLRC may contribute to nucleosome stability indirectly by depositing H3K9me, which prevents access to RNAPII and, thus, nucleosome eviction through transcription. Conversely, stabilized nucleosomes will contribute to maintaining critical levels of H3K9me at TAS. SHREC may contribute to nucleosome stability directly through nucleosome positioning via its ATP-dependent remodeler activity conferred by Mit1; indirectly through histone deacetylation via the HDAC moiety Clr3, thereby preventing open chromatin structure and access to RNAPII
(A) RNA affinity chromatography using the p53 5'UTR (5'p53) or a portion of the p47 5'UTR (5'p47) (depicted in (i)) and HeLa cytoplasmic extracts (CE), followed by western blot analysis (ii).
F Quantitation of FMRpolyG-EGFP band density, normalized to β tubulin band density, from blots in E (Student's t test; n=4-5/genotype). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).
S, T) Quantification (S) and representative gel (T) for inhibition of Ang1-dependent pAKT signaling by Ang2, H3, Tet1-A, or Icos1-A; pAKT band intensities were normalized to Ang1-dependent pAKT level). Representative immunoblots for H3, Tet1-A, and Icos1-A competition experiments are in Appendix Figure S1M-O. Quantification of pERK1/2 and pFAK level for the competition assay is in Appendix Figure S1P-Q. Partial valency analysis of icosahedral nanocages is in Appendix Figure S1R-T.
G A second copy of DDmyc-PKAc1 is stabilised after 1 hour in presence of Shld-1.
Relative mRNA expression of PGC1α and PLIN5 in C2C12 cells transfected with miR-183 and miR-96 agomirs (Ago-183/96) or antagomirs (Ant-183/96) (n=3).
(G) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (C). (H) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (D). ( Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Bars show the fraction (mean ± SEM) of membrane GL and GPL lipids with each fatty acyl chain characteristic. n = 3 MS analyses of 4DO fat body samples for each genotype. MUFA, mono-unsaturated; PUFA, poly-unsaturated. ‡ indicate a significant difference between w- and dTorKO expressing Luciferase RNAi, * indicate a significant difference compared to dTorKO expressing Luciferase RNAi. Two-way ANOVA, Dunnett's multiple comparison test. ‡ p < 0.05 and * p < 0.05.
E. Violin plot showing the RNA expression levels of human LAG3 in human NSC-derived neural cultures. Identities annotate different clusters: Neuronal clusters are comprised of the following markers: GAD2, GABRG1, NTRK2, NEFM, SNCG, SLC17A6, SCN2A, DDIT3/HRK. Mixed glial clusters are defind by the following markers: GFAP, S100B, STMN2, NRN1, GPM6B, COL1A1, with astrocyte-specific clusters characterised by GFAP, S100B, GPM6B, COL1A1. LAG3 cannot be evidenced in any of the clusters beyond few random events. Data shown from 5'476 unique analysed cells from one out of two independent biological replicates.
(F-K) Comparative analysis of GS structures from several organisms. Lateral (F) and top view (G) of the entire GS decameric (a dimer of pentamers) structure are reported in pink cartoon representation. (H) Cartoon representation showing the top view of the monomer-monomer interface hosting cofactors, substrates or inhibitors (Dataset EV1) participating to or inhibiting the conversion of glutamate to glutamine. In particular ADP (cyan), Phosphate ions (brown), MSO phosphate (P3S, yellow), phosphoaminiphosphonic acid-adenylate ester (ANP, white), glutamate (magenta), citrate (black) and the imidazopyridine inhibitor ((4-(6-bromo-3-(butylamino)imidazol(1,2-a)pyridin-2-yl) phenoxy) acetic acid, green) are reported in sticks representation, whereas Mn2+ ions are reported in pale blue spheres and Mg2+ in pale green spheres (involved in the coordination of the imidazopyridine inhibitor). (I) Superimposition of all the sampled 17 crystallized structures (Dataset EV1). (J) 2D representation of glutamate, P3S, glufosinate and glyphosate. (K) Zoomed view of the crystallized P3S binding region and the docked glufosinate and glutamate binding regions within the superimposed human (white cartoon) and Z.mays (pink cartoon) GS structures.
(D) Confocal fluorescence microscopy images and associated quantifications of caspase-11-C254G-GFP (green) recruitment to S.Tm-mCherry (orgA-, red) in IFNγ-primed iWT, iIrgm2-/-, iGBPChr3-/- and iGBPChr3-/-/sgIrgm2 BMDMs after 8 hours of infection. Nucleus (blue) was stained with Hoescht; scale bar 5µm. For quantifications, the percentage of bacteria positive for caspase-11-C254G-GFP was determined by combining the bacterial counts from n=3 independent experiments and expressed as mean ± SEM. ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction.
(E) A cartoon of the molecular details of the ATP-dependent Rad50 dimer-DNA interaction. Schematic illustrations of all direct MjRad50 dimer-DNA interactions show that DNA recognition by Rad50 dimer is symmetrical. The phosphate group is marked with a closed circle. Hydrogen bonds and ion pairs between MjMR and the DNA molecule are indicated by arrows. Bases are numbered as in Fig 1B. Residues that interact with DNA via side-chain are boxed, and via main-chain (Ser109) is shown in two green boxes.
C) Immunoblot showing efficient expression of Miro1 lysine mutants in FlagParkin expressing SH-SY5Y cells.
(C) ZEB1-only peak at the promoter of LLGL2, a well-known ZEB1 repressed target.
(E) SNP array plots showing chromosomal alterations following CEP55 overexpression in MCF10A cells. Red box indicates an extra chromosome 20 in CEP55-overexpressing cells.
(C) Immunoelectron microscopy assay showing that endocytosed CD16:7-263-281 localizes to LC3−labelled, single−membrane vesicles. JAR cells were transfected with the CD16:7-263-281 chimera and a construct expressing human IgG1 fused to LC3A. Cells were subjected to anti−CD16 aggregation for 8 h and processed for immunoelectron microscopy. Thick gold signal (18 nm): aggregated, endocytosed chimera; thin gold signal (12 nm): IgG1-LC3. Arrows indicate single membrane (black) or IgG1-LC3A (white). Scale bar: 400 nm.
(F, G) Quantitative proteomic analysis (iTRAQ) of hindlimb Skm from wt and LowOXPHOS mice. The pie charts (F) and volcano plot (G) present the upregulated (log2[LowOXPHOS/wt]≥1, red) or downregulated (log2[LowOXPHOS/wt]≤-1, blue) proteins. A (-) log p value of >4 was considered statistically significant. Data information: Data are the mean ± s.e.m. of n= 12 animals/ genotype measured in 2 different iTRAQ analysis *p <0.05 when compared to wt by ANOVA and Student's t-test.
D. Control and biotin-treated HEK293T cells stably expressing V5-BirA* and V5-BirA*-AURKA were lysed and biotinylated proteins were precipitated by streptavidin beads. The initial sample (initial) and captured biotinylated proteins (pulldown) were run on an SDS gel and immunoblotted with the fluorescent streptavidin and antibodies against V5 and vinculin (loading control).