EMBO/sd-character-level-ner huggingface dataset:
Quantification of mononuclear phagocytes (Ly6G-, F4/80+, CD11b+) in retinas and sclera-choroid-RPE cell complexes at D3 relative to Naïve; n = 5 (Naïve), 4 (CNV).
(C) Knockdown of SIRT5 leads to higher ROS levels in HEK293T cells. ROS levels were determined in the indicated stable cells as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments ***p<0.001 (two-tailed unpaired t-test).
AREG mRNA levels in HT3 cells incubated for 48 h with or without recombinant HPV E6. Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.0042).
A-B. GABAergic neuron defects in wild-type fish treated with ER/Golgi traffic inhibitor BFA (n=9; ****p<0.0001; ns, not significant; One-way ANOVA).
(C) Overexpression of MIR376A resulted in a decrease in the autophagic activity of MCF-7 cells. Starvation-induced conversion of LC3-I to LC3-II in MCF-7 cells was analyzed. Tests were performed in the presence or absence of E64d (10 µg/ml) and Pepstatin A (10 µg/ml) (E+P). LC3-II/LC3-I densitometric ratios were marked. ACTB was used as a loading control.
Table of the number of spectral counts from proteins with identified diGly- modified peptides, following pulldown by HALO-NEDP1 CA or the HALO-NEDP1 DAGC reduced binder control. NEDD8 is the protein most highly modified by NEDD8.
Puromycin-PLA signal in PMN grown in microfluidics devices. Images are magnifications from the white boxes in Appendix Fig S6 taken from the axonal compartment. The scale bars are 10 µm. White arrows point to puromycin-PLA puncta within the β3-tubulin mask; the empty arrow shows a PLA puncta not associated with neurons.
Each circle represents a distinct protein pair (ortholog or isozyme) between species. Each plot shows identical point values but highlights a different functional class of proteins with relevance to oxidative stress. The y=x diagonal line is a reference to compare intrinsic vulnerability to carbonylation between orthologs. The y=x/3.78 diagonal line is a reference to compare combined intrinsic and extrinsic carbonylation properties between orthologs. Elliptical dotted line encircles the points falling within 3 standard deviations of the mean coordinates and 3 standard deviations of the distance from reference line y=x, encompassing ~91% of all data points. This reference region distinguishes outlier points that are distant from the main population. Outliers with associated experimental evidence related to hypersensitivity to oxidative stress are labeled with their protein names. See also Appendix Fig S3 and Fig EV5.
Brains from young (5 wph) and old (39 wph) were homogenized and clarified lysates separated by size-exclusion chromatography (SEC). For each age group, two pools of brains were processed separately. For each experiment (4 in total), 39 fractions were collected along the chromatogram, digested into peptides and analysed by Data Independent Acquisition (DIA) quantitative mass spectrometry.
Depletion of BmSpnE or BmQin results in segregation of Siwi-D670A from BmDcp2-containing P-bodies. (Top-left) Representative Z-projections (Maximum intensity). Scale bar 8 μm. (Top-right) Enlarged area from the white box. Scale bar 2 μm. (Bottom) Box plot showing that depletion of BmQin (n=32 cells) or BmSpnE (n=32 cells) reduces colocalization ratio between Siwi-D670A and BmDcp2, compared to control (dsRLuc, n=32 cells). Representative data from N = 3 independent experiments are shown. Bonferroni-corrected P-values were calculated by Asymptotic Wilcoxon rank sum test. See also Figure EV3.
G) Hdc inhibition significantly reduced the percentage of Dpn+ NBs (n=16,17), and significantly increased the percentage of Elav+ neurons per clone (n=16,17).
C Frequency of C179+ or V5-tag+ 293T cells 24h after transfection of AAV-vector plasmids as measured by flow cytometry. Symbols represent single experiments and bars the mean ± SE (n = 3).
SUMOylated Euc1 is required to recruit Slx5 and Slx8 to ub-HSs. ChIP against Slx5 in the indicated genetic backgrounds were quantified by qPCR. Note that Slx5 is still recruited in the absence of Slx8 (3HA-Slx5 slx8∆), but not vice versa (Slx8-9myc slx5∆). Data for WT and 3HA-Slx5 in (F) and WT
D. Super-shift EMSA analysis of TFIIA binding to yTBPDNA complex. We incubated 5 nM FAM-labeled DNA and yTBP (0.5 μM) with indicated concentrations of purified TFIIA (0.08 to 5 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).
C Radioactive in vitro AMPylation reactions containing the indicated FICD proteins, [α-32P]-ATP, and BiPT229A-V461F were analysed by SDS-PAGE. The radioactive BiP-AMP signals were detected by autoradiography and proteins were visualised by Coomassie staining of the gel.
J. Average migration speed (expressed as μm/min) of n=30 randomly selected CFP positive cells in intravital images of Control and RASSF1C expressing MDA-MB-231 tumors. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
E Immunostaining analysis of the expression of pluripotency markers (POU5F1, SOX2 and NANOG) in WT and Mettl5 KO mESCs, respectively. DAPI (blue) was used as a nuclear counterstain. Scale bars represent 10 μm.
Immunoprecipitation (IP) with an anti-TRAPPC2 Ab or IgG from cells with or without SA treatment, as indicated, followed by SDS-PAGE and Western blot with ant-Raptor (F) Abs. Input: cell lysate (40 µg for TRAPPC2, 10 µg for Raptor).
D, E, Immunoblots (D) and viability assay (E) of H358 and A549 cells transfected with E2F1-specific or control siRNAs followed by treatment (48 h post transfection) with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536(5 nM) for additional 24 hours. Data were shown as mean ± SD (n=3). **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test.
P-Q. p-ERK is reduced in mutants following treatment with ephedrine as shown by Western blot quantification (N=4). ****p<0.0001, one-way ANOVA.
B Cell morphology using high-power magnification (40x) of the cultured cell lines under a microscope in LCC9 model-9-control and 9C2 cells. Scale bar: 50 µm.
(F) Western blots of protein extracts from MCF-7 transfected with YFP-p27, YFP-p27T198A or YFP-p27T198D mutants following incubation with cycloheximide (CHX) for the indicated times. Uncropped images of the blots are shown in the Supplementary Information, Fig. S5.
HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer. Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- or Lck-Cre;Ptpn2fl/fl;Stat5fl/+ HER-2 CAR-T cells were incubated with plate-bound α-CD3 and CXCR3 MFIs on CD8+CD44hiCD62Llo CAR-T cells determined by flow cytometry.
(C) Effects of Htt-552 on lysosomal stability. The release of β-hexosaminidase from intact lysosomes incubated with Htt-552-18Q or Htt-552-100Q. PC12 cells were transfected with Htt-552 for 48 h. Cell lysates were used as Htt-552-riched lysates. The lysates containing Htt-552 were incubated with isolated lysosomes and the lysosomal β-hexosaminidase activity at time 0, 10, 20 and 30 min was determined. Release of all lysosomal β-hexosaminidase from the same batches of lysosomes was induced by incubation with 0.1% Triton X-100. Release of all lysosomal β-hexosaminidase from the same batches of lysosomes was induced by incubation with 0.1% Triton X-100. The values are means±SE of three independent experiments.
A. Histograms of spindle angle of GFP-H2B U2OS cells transfected with either GAPDH or Spindly siRNA and filmed on rectangular micropatterns. Cells were filmed 48 hours post-transfection.
(C) Representative bright field images of migrating Daoy-SOX9/T-FBW7α in the absence or presence of Dox. Daoy-SOX9/T-FBW7α cells were either maintain in regular culture or pre-conditioned with dox prior to experiments. The cells (2 x 105cells / insert) were seeded and let to migrate across 5 μm filter pore toward the laminin-coated trans-well for 4 hours prior to fixation and staining with crystal violet. Histogram quantification of cell migration were performed by measuring the crystal violet absorbance of the stained-migrating cells and presented as mean + standard deviation from 2 independent experiments each containing 2 technical replicates.
C to E Clinical score (C), body temperature (D), and relative weight (E) during challenge period. Statistical significance between pre-immune and immune sera was determined using Friedman test with Dunn's multiple comparison testing (AAV-HA ##p<0.01; AAV-cHA §§p<0.01; AAV-GFP $p<0.05; $$p<0.01; QIV *p<0.05;). Mean ± SD
E, F. In vitro T cell suppression activity of CT26-tumor infiltrating CD11b+ myeloid cells collected at 2 weeks post-treatment with vehicle or 75 mg/kg TP-16. CD8+ T cells were labeled with CFSE and then stimulated with CD3/CD28 antibodies for 3 days in the absence or presence of tumor-infiltrating CD11b+ myeloid cells from vehicle- or 75 mg/kg TP-16-treated CT26 tumors. Representative flow cytometry histograms (E) and percentages (F) of proliferating CD8+ T cells when plated in a ratio of 2:1, 1:1, and 1:2 CD8+ T cells to CD11b+ myeloid cells (n = 3).
(b) MEFs as in a but expressing EGFP-Parkin were treated with CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis. The asterisk indicates a nonspecific band.
(C) Distribution of LAMP‐2A (L‐2A) and flotillin‐1 (Flot‐1) in fed rats liver lysosomes extracted with 1% Triton X‐114 at 4 or 37°C. DR fractions were pooled as in (B), and the aliquots below this region were divided in three fractions (a soluble (Sol; aliquot 15) and two intermediate fractions (light: aliquots 11-12; heavy: aliquots 13-14).
(B) The cleavage is efficiently inhibited by 1 μM STS736, a specific peptidyl ketoamide rhomboid inhibitor Ticha et al., 2017b(). Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE) and ectopic YqgP by anti-YqgP antibodies.
B) Representation of glutamylation patterns found in the knockout mouse models studied here, and interpretation of the participation of the TTLL1, TTLL7 and CCP1 in their generation. Based on the immunoblot analysis shown in Fig. 2C, it can be deduced that TTLL1 is able to hyperglutamylate both, α- and β-tubulin in the absence of CCP1. By contrast, loss of CCP1 in the absence of TTLL1 does not lead to hyperglutamylation, suggesting that TTLL7 is not sufficient to generate this phenotype. In this context, our observation that hyperglutamylation of β-tubulin is abolished with TTLL7 indicates that while TTLL1 can hyperglutamylated β-tubulin, it requires prior glutamylation by TTLL7 to do so. We thus conclude that TTLL1 specifically elongates nascent glutamate chains generated by TTLL7.
C. Live cellimages and histograms of GFP-H2B U2OS cells Mock transfected or with siPBIP1 filmed on rectangular micropatterns in the presence of a CENP-E inhibitor. The spindle angles for both conditions were scored at 32 minutes after NEB.
B Hematoxylin and eosin staining (H&amp;amp;E) of skeletal muscle from patient II.1 shows histological features of moderate to severe dystrophic pattern. (Scale bar, 50 μm).
Dimer of full-length cGMP-bound Bd1971; one monomer coloured individually by subdomain, the opposing monomer coloured white. Ligand represented in stick form, EAL active site Mg2+ as green sphere. View from top of that in (A), detailing EAL region of dimer interface. View from opposing side, detailing C-helix dimer interface at cNMP domain. The Bd1971 EAL dimerization interface (purple, white) matches that of a canonical, active dimer from the well-characterized T. denitrificans EAL (tan, PDB 2r6o); the catalytic Asp pairs of the two enzymes adopt a similar conformation (stick form, D306/307 from Bd1971, D646/647 from 2r6o). Structure of the cyclic-di-GMP bound complex of Bd1971 (ligand C represented in yellow, bound Ca2+ ions as green spheres). Residues R182, R330 and Y385 display the largest shift between the apoenzyme (tan) and bound (blue) states, R182 complexing the cyclic-di-GMP phosphate, and Y385 stacking with a guanine base.
Proliferation assay in VSMCs transduced with the indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cells were collected and counted at each time point as indicated. **P<0.01 vs LacZ, by ANOVA with Sidak's correction.
(c) Survival rate of two different miR-212/132 TG mouse families (TG-Fam23, TG-Fam43) versus WT controls was analysed by Kaplan-Meier survival assay (n=87, 65 and 53 for WT, TG-Fam23 and TG-Fam43, respectively).
A, B. RT-qPCR analysis of Zfp207 and Oct4 in mouse ESCs during (A) retinoic acid (RA)-induced and (B) embryoid body (EB)-mediated differentiation. Nestin was used as a neuronal differentiation marker to monitor RA-mediated differentiation. mRNA levels are relative to the expression at day 0.
E-F Human moDCs described in (D) were analyzed for mean fluorescence intensity of CD80 or CD86 by flow cytometry (E) and CCL2 or IL-23 protein by ELISA (F). (n = 7; 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by paired, two-tailed t-test E and F). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. See Appendix Table S3 for exact P‐values.
Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT and KO crypts (E) treated with DMSO or TCDD, n=3 or 4 per group.
(A) PCoA of Bray-Curtis dissimilarity for all (n=221) samples shows that gut communities cluster by mothers and infants.
E Schematic structure of the humanPOGLUT1protein, which contains a signal peptide (SP), a CAP10 domain, and a Lys-Asp-Glu-Leu (KDEL)-like endoplasmic reticulum retention signal. D233E is located in the CAP10 domain (arrow), close to the predicted ERD catalytic motif (asterisk). Alignment of amino acids flanking the mutated aspartic acid from POGLUT1 orthologs indicates evolutionary conservation of D233 in vertebrates (open arrowhead). Black boxes denote similarity of amino acid residues.
HEK293 cells were transfected with empty pcDNA3 vector (-) or Flag-DDX5 (+). Whole cell lysates (WCE) and anti-Flag immunoprecipitations (IP) were immunoblotted with anti-Flag and anti-XRN2 antibodies.
(i) Pseudo-color look-up table applied to mean nuclear Venus::HES5 intensity in (h) (only 1 side of ventricle) after radial gradient removal (see Materials and Methods).
Gene ontology for genes that are two-fold misregulated 5hmC and expression between WT and Kdm3b-KO on day 10.
Dexamethasone treatment accelerates increase in activity of G trehalase and H arginase reaching adult organoid level ( ) at day 13 of the culture. Activity is given in µM glucose/µg protein·min-1 (n = 3 independent organoid cultures).
A Protein expression of SQLE in human liver tumors (C) and non-cancerous adjacent tissue (N) were analyzed by western blotting.
Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing TEAD1 truncation mutants and HOXA4. Arrowheads indicate IgG heavy chain. Arrows indicate TEAD1 (a.a. 1-426).
Left, localization of site 5 at the interface between the two constitutive ABD dimers. Right top, crystal structures of the inhibited (PDB 5IOV; Zhu et al, 2016) and presumably active (5FXG; Tajima et al, 2016) states illustrating the differences in distances between GluN2B-L795 and GluN1-E698 or GluN1-R673 at interface 5. Those three residues are colored green, while GluN1 and GluN2B subunits are colored red and blue respectively. Right bottom, schematic representation of the GluN1-R673C/GluN2B-L795C disulfide cross-link capturing an inhibited state of the receptor, and of the GluN1-E698C/GluN2B-L795C disulfide cross-link trapping the rolling motion
(C) Complex II-III: Succinate dehydeogenase, cytochome c reductase activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).
D. Tace activity was measured from lysates of Niacin-treated cultures, n=7 (KO NT) and n=8 (KO 5mM Niacin) number of independent plates, each containing from 10-15 DRGs plated. Note that Tace activity was similar between WT and Mtmr2-/- cultures, one-tailed Mann Whitney U test, p=0.037.
B, Average ΔF/F0 SyGCaMP5 traces from n=11 neurons (98 terminals) plotting the average of terminals without a mitochondrion (black trace) and with a mitochondrion (red trace). Stimulation occurred for 10s (t=20-30) at 10 Hz.
B. Relative miR-34c-5p and miR-155-5p expression levels (2deltaCt normalized to RNU48) in unstimulated and stimulated naive CD4 T cells of individual samples pooled to generate the sequencing libraries. mean ± SEM and p-value for differences in miR-155-5p expression (paired two tailed t-test) are shown. Comparison could not be performed for miR-34c-5p as it is undetermined in unstimulated samples.
G) relative miRNAs expression levels (G) were quantified in therapy-naïve (N) and resistant (R) PDX samples. The log2 fold change or the ratio of the fold change R vs N is shown for each couple of samples.
(H) The above Dps were used for H&E or Immunohistochemistry (IHC) staining for MyHC. Representative images from 3 pairs of littermates are shown. Scale bar = 50 µm.
MIR376 family miRNAs in the DLKI/GTL2 gene cluster. (A) Individual miRNA genes were indicated. Sizes of spacer sequences between miRNA genes were marked as nucleotides (nt). (B) Comparison of mature miRNA sequences with identical "seed sequences" (bold, underlined). Identical nucleotides were indicated by asterix (*). ClustalW tool was used for the analysis. (C) ClustalW comparison of the stem-loop sequences of MIR376A and MIR376B. Seed sequences were marked as bold and underlined.
E. Results of the phospho-Y2H screen to identify kinases modulating the interaction of AAR2 and PRP8. Growth on selective media (SD5), of strains carrying AAR2 bait and PRP8 prey plasmids is reduced through co-expression of either SGK2 or CK2α1(CSNK2A1). Growth reduction is visible in a 1:10 dilution series on selective agar (SD5) in comparison to the control media (SD3). Ctrl: non-related kinases.
Western blot analysis of the indicated FAO enzymes in melanoma cells treated, or not, with EV from lean (ND) and obese (HFD) mice. Tubulin (TUB) is used as a loading control.
A. Estimated number of molecules and molarity of PBP1a/LpoA and EPases. 1Numbers obtained by ribosomal profiling in rich growth medium (Li et al., 2014). 2Concentration of monomer. 3Decreases in the presence of NlpI (Singh et al., 2015). 4"no change detected". The periplasmic concentrations of proteins were estimated for a cell with periplasmic volume of 3.33 ×10-16 L, where 1 molecule corresponds to 5 nM.
(g) Two-day co-cultures of HeLa cells expressing either MKLP1-GFP (MBd marker) or cytosolic RFP. Green MBds (arrows) associated with red cells (asterisk) indicate post-mitotic transfer of MBds between cells. Scale bar, 10 μm.
In silico lineage tracing of fibroblasts. (E) Labelling of fibroblasts close to the epidermis at indicated MCS. (F) Labelling of fibroblasts in the middle of the dermis at indicated MCS. (G) Labelling of fibroblasts close to the inside of the body at indicated MCS.
(A) Immunoblotting analysis for MIG-6 expression in BT549 cells under normoxia and hypoxia.
M1-linked tetraUb hydrolysis by recombinant OTULINWT and OTULING281R using the indicated OTULIN concentrations and visualised on silver-stained SDS-PAGE gels. Data are representative of three independent experiments.
(A-D) Cluster analysis of proteins enriched at telomeres lacking POT1 using STRING. The largest identified clusters are colored in red (mitosis-associated proteins, (A), green (recombination and DNA damage response proteins, (B), purple (RNA-binding proteins, (C), and dark blue (chromatin and DNA remodelers, (D). Line thickness indicates the strength of data support.
(F) Markers of 5C, naïve and primed states (Dataset EV4) were used to analyze expression profiles of epiblast during the early development of mouse embryos (Data ref: Mohammed et al, 2017a) (F). Single-cell was plotted based on the average expression of each group of markers.
A) Capture-C from hypoxicMCF-7 cells (top) and ChIP-seq tracks for subunits of HIF, H3K4me1, H3K27ac, CTCF and RNAseq in normoxic and hypoxicMCF-7 cells as indicated at the ARRDC3 (arrestin domain containing 3) and LUCAT1 (lung cancer associated transcript 1 - non-protein coding) gene loci. RNA-seq tracks are shown in both low-scale and high-scale to represent regulation of transcripts with differing transcript levels. Capture-C was performed using oligonucleotides to a 'viewpoint' region at the HIF-binding site (highlighted in red). Interaction with the TSS of both LUCAT1 and ARRDC3 is highlighted in blue. Association with regions including additional enhancers, are highlighted in green.B) Regions associated with HIF-binding sites were classified according to the presence of a UCSC annotated TSS, or coincidence with H3K4me1 or H3K4me3 ChIP-seq signal. The bar chart shows the average number of each type of site that interacted per HIF-binding site.C) Histogram showing the frequency distribution of elements interacting with HIF-binding sites according to absolute distance from the 'viewpoint' site on the horizontal axis.
C, ColE9 residues 45-67 (orange) encompassing OBS2, traverse the pore of OmpF subunit 2 (grey). After passing through the eyelet, OBS2 tracks more centrally in the inner vestibule unlike in panel c where residues 3-24 trail the side of the pore.
(c) MEFs stably expressing FLAG-Parkin and either EGFP-FKBP38 or EGFP-FKBP38(N402K) were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis. (d) Densitometric quantitation of EGFP fusion protein abundance in immunoblots similar to those in (c). Data are expressed relative to the corresponding control value (CCCP 0 h) and are means±s.d., from three independent experiments. **P0.01 (Student's t test).
Schemes showing ATG4C (A) and (B) BECN1 MRE (top), or their mutated versions (bottom) placed in the 3' UTR of the luciferase gene in the pGL3 vector. Mutated residues were italicized. MIR376A seed target sequence was underlined.
(H) Representative immunoblots for FoxM1 and PLK1 protein derived from organoids treated with DMSO or TCDD from n=6 independent samples. Total protein was used as a loading control.
(G) Human choroidal EC (HCEC) and mouse retinal EC (MREC) were treated with LIF 10 ng, LIF 100 ng for 30 min. (PBS was used as control). STAT3 phosphorylation was detected by western blot.
H. Representative immunofluorescence staining of CLIMP-63 of femur growth plate sections from mice with indicated genotypes. Scale bar 10μm. Insets showed increased CLIMP-63 staining in Fgfr3/4 dKO mice. Scale bar 5 μm. Hypert= hypertrophic chondrocytes; pre-hypert= pre-hypertrophic chondrocytes; prolif = proliferating chondrocytes.
E The repressive activity of L3MBTL3 at Notch target genes is RBPJ-dependent. Expression analysis of Notch target genes upon RBPJ knockdown and/or overexpression of L3MBTL3. Shown are means ±s.d. of triplicate experiments. P values were estimated via a one-way ANOVA model on log-transformed data where the difference of differences was tested, which is equivalent to testing the interaction in a two-way ANOVA model. Western blot analysis validates the overexpression of L3MBTL3 and the shRNA-mediated depletion of RBPJ (Appendix Fig S3E). Gene expression analyses of OCT4 was performed as control (Appendix Fig S3F).