EMBO/sd-character-level-ner huggingface dataset:
Figure 4a and b, Schematic representation of the potential acetylation domain organization of BRD4 and its lysine mutants (AC1‒AC4). c, Recombinant PCAF acetylates His6-BRD4 at lysine residue 332. Acetylated BRD4 was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times. d, The mCherry-tagged WT and BRD4 mutants were detected in anti-GFP immunoprecipitates by Western blotting in A549 cells. Data information: Each experiment was repeated at least three times. e and f, The mRNA levels of TWIST1(e) and Snail1(f) were verified in A549 cells co-expressing mCherry-tagged WT or mutant BRD4 and GFP-tagged ISX by RT-PCR. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times. g and h, The DNA binding activity of TWIST1 and Snail1 were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190/+40 bp, negative control) (10% input of each groups were pulled down and used for qPCR analysis). Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times. i, The cell invasion (transwell) activity were determined in A549 cells co-transfected with cDNA coding for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.
Increased sensitivity to hallucinogenic drugs in the Ldb2 KO mice A, B.Figure 3Locomotor activity was assessed in the same experimental set as in the open-field test. After acclimation for 120 min, mice (n = 10-12/group) were injected (arrows) with methylphenidate (MPD: 3 mg/kg, A) and their locomotor activity was monitored. In the right panels, cumulative values for pre- and post-injection times are also shown. Locomotor activity was assessed in the same experimental set as in the open-field test. After acclimation for 120 min, mice (n = 10-12/group) were injected (arrows) with MK-801 (0.2 mg/kg, B) and their locomotor activity was monitored. In the right panels, cumulative values for pre- and post-injection times are also shown. C. Popping behavior induced by the injection of MK-801 (0.5 mg/kg) was manually assessed in WT (n = 4) and KO (n = 6) animals. Typically, each episode is constituted by blocks of 5-15 successive jumping behaviors. Episode numbers in 20 min (left) and total duration (right) are shown in a box-whisker plot.
Figure 1Western blot analysis and quantitation of Sel1L-Hrd1 ERAD proteins in the livers of WT mice at 3, 9 and 24 weeks of age (n=3-6 per group, 2 independent repeats). α-tubulin, loading controls for Western blot analysis. Western blot analysis and quantitation of Sel1L-Hrd1 ERAD proteins in the livers of 10-week-old WT mice under fasted (overnight) or ad libitum fed conditions (n=3-6 per group, 2 independent repeats). α-tubulin, loading controls for Western blot analysis. Western blot analysis and quantitation of ERAD proteins in Sel1Lf/f and Sel1LAlb livers (n=4 per group, 3 independent repeats). Hsp90 loading controls for Western blot analysis. Growth curves of male (n=10 each) and female (n=7 each) mice. Representative images (E) and nose-to-anus length (F) of male mice at 6 weeks of age (n=6-10 per group). Organ-to-body-weight ratios of liver and kidney in 6-week-old male mice (n=6 per group, 3 independent repeats). Daily food intake (g/d) normalized to gram of body weight (gbw) (n=3 per group, measured over 3 days). Representative estrus cycle mapping in 2-4-month old females (n=6 per group). H&E images of paraffin-embedded liver sections from 9-week-old mice (n=4 per group, 3 independent repeats). Western blot analysis of cell death (cleaved and pro-caspase-3) in livers of 9-week-old mice, with WT ileum as positive control (n=3 per group, 3 independent repeats). Hsp90 loading controls for Western blot analysis. Representative transmission electron microscope (TEM) images obtained from 9-week-old female mice livers (n=10-12 cells from one mouse each). N, nucleus; mito, mitochondria; ER, endoplasmic reticulum.
Analysis of autophagy in trisomic and tetrasomic cell lines.Figure 6(A) Tri‐ and tetrasomic cells accumulate p62‐positive foci. This effect was further increased by treatment with Bafilomycin A1. Right panel: Quantification of the fluorescence intensity changes; significance was evaluated by non‐parametric T‐test (***P<0.001), '+' designates addition of Bafilomycin A1. (B) p62‐positive foci co‐localize with ubiquitin‐positive foci and their fluorescence intensity is increased in aneuploid cell lines. Plots represent the signal intensity along the indicated gray line. Red line-p62, green line-ubiquitin. Source data is available for this figure in the Supplementary Information.
a, b, LDH release, IL-1&bgr secretion (a) and immunoblots for caspase-1, caspase-11, IL-1&bgr, IL-18 and IL-1&agr (b) from unprimed BMDMs infected for 16 h with the indicated bacteria (grown to stationary phase).f1a, b, LDH release, IL-1β secretion (a) and immunoblots for caspase-1, caspase-11, IL-1β, IL-18 and IL-1α(b) and immunoblots for caspase-1, caspase-11, IL-1β, IL-18 and IL-1α (b) from unprimed BMDMs infected for 16h with the indicated bacteria (grown to stationary phase). c, Time course measuring LDH release from unprimed or IFN-γ-primed BMDMs infected with S. typhimurium d, e, LDH release and IL-1β secretion from primed BMDMs infected with SPI-1-expressing logarithmic phase S. typhimurium, treated with monosodium urate, alum and nigericin or transfected with poly(dA:dT) and LPS. LDH release and IL-1β secretion from unprimed wild-type and Gbp2−/− BMDMs infected for 16h with the indicated bacteria (grown to stationary phase). Graphs show mean and s.d. of quadruplicate wells and data are representative of two (b) and three (a, c-f) independent experiments. *Crossreactive band; **P<0.01; NS, not significant (two-tailed t-test).
The centrosome regulates MT nucleation activity of the GA A Control and centrinone-treated cells were subjected to a 3-min MT regrowth assay, fixed and labelled for EB1 (green) and giantin (red).Figure 4Control and centrinone-treated cells were subjected to a 3-min MT regrowth assay, fixed and labelled for EB1 (green) and giantin (red). Insets show magnified images of boxed regions. Workflow of the method used for quantifying MT nucleation from the GA. Quantification of the total number of growing MTs per cell (left), the number of MTs nucleated per Golgi fragment (middle) and the number of MT-nucleating Golgi fragments (right) in both control and centrosome-less RPE-1 cells. Scatter plots show each individual data point. Horizontal black lines bars represent the mean. RPE-1 cells were treated wi­­th centrinone for either 7 days (left) or for 5 days and then allowed to recover for 48h in the absence of the drug prior to fixation (right). Representative images of a triple IF staining with EB1 (green), γ-tubulin (red) and giantin (blue) as a Golgi marker are shown. Insets show MT nucleation from Golgi elements (left) or from multiple centrioles (right) and are enlarged at right. Arrowheads indicate the position of Golgi elements. Quantification of the percentage of cells exhibiting Golgi-nucleating activity in centrinone-treated cells 48h after the washout of the drug. Cells were divided into two categories depending on the number of centrioles that were visualized by labelling for the centriole protein Cep63. At least 100 cells from two independent experiments were analyzed in each condition.
Figure 5(A) Histological analysis of plaque-associated BACE1 immunoreactivity in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. BACE1 area covered was normalised to 4G8-positive area covered of the same image (left). Right: representative images, scale bar = 50 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.1780. (B) Stereological quantification of the number of cortical GFAP-positive astrocytes in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice (left). Right: representative images of GFAP staining, scale bar = 200 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.3148. (C) Quantification of activated microglia within 30 µm from plaque borders. Top: Numbers of Iba1-positive microglia were normalised to the size of the nearest 4G8-positive plaque and quantified in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.1925. Bottom: Histogram representing Clec7a staining intensity within plaque-associated Iba1-positive microglia in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for each single bin, p=N.S.. Right: representative images, scale bar = 40 µm. (D) Radial intensity profiles of Iba1 and 4G8 around the center of the nucleus of plaque-associated Iba1-positive microglia in male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. Iba1 intensity declines until a radius of ~6 µm, marking the cell periphery. 4G8 intensity peaks inside the cell (~4 µm), but stays high outside the cell. This is very likely due to the close proximity to 4G8 positive plaques. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test with Bonferroni correction for the number of binned radii shows no significant difference between both 4G8 traces.
Figure 1Schematic representation of the domain structure of METTL14. The C-terminal IDR, containing an array of RGG motifs, is highlighted. NHM: N-terminal α-helical motif; MTase: Methyltransferase domain; IDR: Intrinsically disordered region. METTL14 is arginine methylated in vitro. In vitro methylation assays were performed by incubating recombinant PRMTs (1-8) with purified GST-tagged METTL14. Ponceau staining shows the loading of the recombinant proteins. The black dots indicate PRMT enzymes; black triangles indicate arginine methylated-METTL14; open triangles indicate recombinant METTL14 proteins. METTL14 is arginine methylated at its C-terminal IDR in vitro. GST-tagged full-length (FL) and C-terminal IDR-truncated (1-400) METTL14 were incubated with recombinant PRMT1 and PRMT6. Ponceau staining shows the loading of the recombinant proteins. The black dots indicate PRMT enzymes; black triangles indicate arginine methylated-METTL14; open triangles indicate recombinant METTL14 proteins. METTL14 is arginine methylated in cells. Endogenous METTL14 was immunoprecipitated from HEK293 cells under denaturing conditions and detected using the ADMA antibody ASYM26. the black triangles indicate arginine methylated-METTL14; open triangles indicate the immunoprecipitated METTL14 proteins. Inhibiting type I PRMT activity reduces METTL14 arginine methylation. HEK293 cells were treated with the type I PRMT inhibitor MS023 (1 μM, 48 h). METTL14 was immunoprecipitated from the cells and detected by Western blot analysis using anti-METTL14 and anti-ASYM26 antibodies. the black triangles indicate arginine methylated-METTL14; open triangles indicate the immunoprecipitated METTL14 proteins. METTL14 is arginine methylated at its C-terminal IDR in cells. HEK293 cells expressing Flag-tagged FL or C-terminal IDR-truncated (1-400) METTL14 were lysed and immunoprecipitated with an anti-Flag antibody. Arginine methylation of immunoprecipitated METTL14 was analyzed by Western blot using two different antibodies that recognize ADMA. the black triangles indicate arginine methylated-METTL14; open triangles indicate the immunoprecipitated METTL14 proteins.
in human airway basal progenitor-like cells and lung cancer.Figure 5(a-d) BCi-NS1.1 cells were stably infected with pLKO control or shEMP1.1. Data are representative of n=3 independent experiments. (a) Total RNA was isolated and analysed for EMP1 expression using Taqman/qPCR with a GAPDH control. Error bars denote mean ± SEM, ***, p = 0.0003. (a-d) BCi-NS1.1 cells were stably infected with pLKO control or shEMP1.1. Data are representative of n=3 independent experiments. (b) Filter grown cells were fixed and stained for ZO-1 and DNA and analyzed by confocal imaging. (a-d) BCi-NS1.1 cells were stably infected with pLKO control or shEMP1.1. Data are representative of n=3 independent experiments. (c) Quantification of tight junction phenotype. >500 cells were counted per sample/experiment, across n=3 independent experiments (dots indicate individual data points). Error bars denote mean ± SEM. *, p = 0.0276. (a-d) BCi-NS1.1 cells were stably infected with pLKO control or shEMP1.1. Data are representative of n=3 independent experiments. (d) Cells were cultured on filters under ALI conditions and transepithelial resistance (TER) was measured on day 21 post-seeding. Error bars denote mean ± SEM. ****, p < 0.0001. (e) EMP1 mRNA expression in normal lung versus cancer tissue. The Hou Lung dataset was analyzed using Oncomine software and the 213895_at reporter [45]. Box whisker plots are presented. The points represent the minimum/maximum values, bars denote the 10th-90th percentiles, boxes show the 25th-75th percentiles and the horizontal line marks the median. The number of samples analysed (n), the fold-change (bold) and the p-value are indicated.
Figure 1A Jurkat T cells (WT), ARHGAP45-deficient Jurkat T cells (ARHGAP45-/-) and 5 independent clones (C1 to C5) of Jurkat T cells expressing ARHGAP45OST molecules were analyzed for ARHGAP45 protein expression. Immunoblot analysis of equal amounts of proteins from cell lysates that were either directly analyzed (Total lysates), or subjected to affinity purification on Strep-Tactin Sepharose beads followed by elution of proteins with D-biotin (Affinity purification), and probed with antibody to ARGHAP45 or anti-HSP60 (loading control). Left margin, molecular size in kilodaltons (kDa). B Volcano plot showing proteins enrichment (fold change in log10 scale) after affinity purification in Jurkat T cells expressing ARHGAP45OST molecules compared to affinity purification in control Jurkat T cells expressing similar levels of WT (untagged) ARHGAP45 proteins prior to (unstimulated) and at 120 s after (stimulated) TCR stimulation. ARHGAP45 interacting proteins with a greater than 2-fold enrichment and a p-value < 0.01 were selected as specific ARHGAP45 interactors (Dataset EV1) and some of them are specified in red. Red lines represent the thresholds set on P-value and enrichment to identify specific ARHGAP45 interactors. C ARHGAP45-specific interactors highlighted in the text are displayed in a stoichiometry plot (Voisinne et al., 2019) where the ratios of the prey to bait cellular abundances ('abundance stoichiometry') are plotted as a function of their maximal interaction stoichiometries ('interaction stoichiometry'), both using log10 scale (see Datasets EV1 and EV2). The ARHGAP45 bait is shown as a yellow dot. The size of the dots is commensurate to the maximal protein enrichment in ARHGAP45OST samples as compared to WT control samples. The TNKS prey for which it was not possible to determine the cellular abundance is shown at the bottom of the stoichiometry plot. For each time point, three independent biological replicates were performed and each biological replicate was analyzed in triplicate by MS.
Migration of transient definitive EMP from yolk sac to fetal liver and accompanied differentiation to fetal liver monocytes increases MΦTR developmental potential.Figure 1Representative flow cytometric analysis of BAL from Csf2ra-/- recipients 8 weeks after neonatal i.n. transfer of YS EMP from CD45.1+ E10.5 embryos, 8-week old un-transferred Csf2ra-/-, and WT (CD45.2) mice. Pre-gated viable CD45+ singlets. Numbers of EMP effectively transferred in neonates (white circle) and numbers of EMP-derived AM (red circles) in BAL and lung of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Numbers of EMP-derived AM in the BAL (left) and lung (right) (C) in the BAL of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls. total protein in the BAL of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls. total eFluor780+ events (E) in the BAL of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls. Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) in the BAL and lung from Csf2ra-/- recipients 8 weeks after neonatal transfer of 1:1:1 mixture with E17.5 fetal liver (FeLi) monocytes (Mo) (CD45.1+), E14.5 FeLi Mo (CD45.1+CD45.2+), and E10.5 YS EMP (CD45.2+) (F) or E17.5 fetal lung (FeLu) Mo (CD45.1+), E14.5 FeLu Mo (CD45.1+CD45.2+), and E10.5 YS EMP (CD45.2+) (G) (n = 3 mice).
Figure 3A Nissl stained slice (left) and corresponding section from brain atlas (right). Left panel shows hippocampus and position of the stimulating (left) and recording (right) electrode. Scale bars: 500 µm (black bar). B LTP protocol was applied to 16 slices of Gadd45a-WT mice (white dots, n=5 animals) and 11 slices of Gadd45a-KO mice (blue dots, n=5 animals), resulting in a significantly (p<0.05, Student t-test) lowered induction of LTP in Gadd45a-KO mice as compared with Gadd45a-WT. Values shown are mean ± SEM. C Gadd45a-overexpressing mice (red dots, n=10 slices from 6 animals) presented a slightly increased LTP as compared with Gadd45a-WT (white dots, n=8 slices from 6 animals), which became more pronounced during the late phase of the experiment. Values shown are mean ± SEM.
Figure 5Nude mice (nu/nu) were injected subcutaneously with 5 x 106 human DLD1 cells, BRCA2-proficient (A) or BRCA2-deficient (B). Tumour-bearing mice were treated with 3 mg/kg daily chlorambucil administered intraperitoneally (i.p.) for a total of ten days. Tumour weight was determined on the indicated days after initiation of the treatment. PDTCs derived from breast cancer samples as previously described (Bruna et al, 2016) were treated with chlorambucil at the indicated doses. Cell survival is represented relative to DMSO control. AB521, ER-negative tumour, no BRCA1 alteration; STG201, tumour with BRCA1 promoter methylation and loss of BRCA1 expression; VHIO179, tumour with BRCA1 germline mutation and MAD2L2 inactivating mutation (olaparib-resistant) CB17/SCID mice were injected intramuscularly with 5 x 106 human BRCA2-deficient HCT116 cells. Tumour-bearing mice were treated on the indicated days with chlorambucil or cisplatin administered intraperitoneally (i.p.), or talazoparib administered orally (o.s.) Tumour volume was measured on the indicated days after treatment initiation and was expressed relative to tumour volume at the beginning of treatment (day 1). Scale bar, 40 µm. Each experimental group included n = 5 mice. Error bars represent SEM. Tumour sections were assessed at the end of each treatment using immunohistochemistry of γH2AX staining.
Figure 8(A) Western blot analysis PABPDHFR cells depleted of LSM1 by siRNA-mediated knockdown. Transfected cells were subsequently cultured in the presence or absence of TMP to maintain or deplete endogenous PABP. (B) PABPDHFR cells were transfected with siRNAs targeting GFP (control) or LSM1. 48 hours later, transfected cells were split and cultured in the presence or absence of TMP for 12 hours. The stabilities of select mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. (C and D) Poly(A) tail analysis of GAPDH and ACTB mRNAs from total RNA isolated from PABPDHFR cells described in (B) was determined by extension poly(A) tail (ePAT). PCR products were run on high-resolution agarose gels. 'A12' represents the size of ePAT RT-PCR amplicons derived from a mRNA with a fixed (A12)-tail.
The LSD endolysosomal membrane contains increased amount of cholesterol‐enriched regions.Figure 4(A) Endolysosomal membranes from MSD, MPS‐IIIA and WT MEFs were treated with 1% Triton X‐114 and loaded on a sucrose gradient. Immunoblots with Flotillin‐1 identified DRMs in fractions 2, 3 and 4 (arrows). The fractions at the bottom of the gradient (12 and 13) correspond to high‐density detergent soluble fractions, whereas the remaining ones were defined as intermediate fractions (intermediate‐I: 5, 6, 7 8; intermediate‐II: 9, 10 and 11). The percentage of Flotillin‐1 in DRMs was calculated from the densitometric quantification of immunoblots. (B) Immuno‐EM of GM1 lipid was carried out in WT, MSD and MPS‐IIIA MEFs by staining cells with anti‐cholera toxin B antibodies (see Materials and methods section). The number of GM1‐positive dots was measured in 25 cells from three independent experiments and displayed as fold to WT. (C) Endolysosomal membranes from MSD, MPS‐IIIA and WT MEFs were stained with C‐laurdan and subsequently analysed by fluorescence spectrophotometry to calculate the GP value (see Materials and methods section for details). Distribution of cholesterol was also measured throughout the gradient ad expressed as percentage of total cholesterol in raft (DRMs) and soluble fractions. (D) Equal aliquots from either DRMs or soluble fractions were pooled, the protein content determined and displayed as percentage of total protein in DRM and soluble gradient regions. (E) Immunoblotting profiles of the transferrin receptor and LAMP1 in the sucrose gradient. Values represent the mean±s.e.m. values of three experiments (A-D). *P0.05, Student's t‐test: (A-C): WT versus MSD and WT versus MPS‐IIIA; (D): WT versus MSD and WT versus MPS‐IIIA for each fraction. Scale bar: 0.3 μm (B).
Figure 6A YOD1 can link p97 to ubiquitin. Ubiquitin-GST (Ub-GST) or GST alone was incubated with the indicated purified proteins and isolated. Co-isolated proteins (pulldown) were detected in SDS-gels. Note binding of p97 and YOD1 to Ub-GST only in combination. B The S2 site of YOD1 is critical for ubiquitin binding. Experiments as in (A) with indicated combinations of p97 and YOD1-wt or the ubiquitin-binding mutant, MutS2. C Reconstitution of a complex of ELDR components on ubiquitin. Experiments as in (A) with combinations of p97-wt or p97-EQ, YOD1, PLAA and UBXD1 as indicated. Note that PLAA associates with p97 and YOD1 on ubiquitin only in the case of p97-EQ. D Translocation of YOD1 to lysosomes depends on ubiquitin binding by the S2 site. LLOMe-treated cells expressing GFP-YOD1-CS or the double mutant GFP-YOD1-CS-MutS2 were chased for 2 h and stained for K48 chains and LAMP1. Ratio of GFP signal on K48 vesicles to GFP signal in the whole cell was determined. E The S2 site is essential for K48-conjugate removal. GFP-YOD1-wt or GFP-YOD1-MutS2 overexpressing cells treated as in (D) were stained for K48 chains. Quantification of the number of K48-positive vesicles per cell. Data represent mean ± SD of three independent experiments. ** = p < 0.01 (Student's unpaired t-test). Scale bars, 10 µm.