EMBO/sd-character-level-ner huggingface dataset:
Impact of the α1S subunit knockdown in maintenance of muscle ultrastructure.Figure 5(A) Ultrathin sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were imaged by electron microscopy. Cis, terminal cisternae; T, tubule; SR, sarcoplasmic reticulum. Bars: 500 nm. (B) Myofibres isolated from Ctrl or ΔDHPR FDB muscles 6 months post injection were processed for immuno‐fluorescent labelling for RyR1 (green), SERCA (red), Dapi (blue) and imaged by confocal microscopy. Scale bars, 10 μm.
Pacs1 deletion induces ER stress, ROS, and heightened sensitivity to oxidative stress.Figure 4(A) Immunoblot of ER mass, ER stress, and autophagy markers in Pacs1+/+ and Pacs1-/- splenic B cells that were left unstimulated or stimulated overnight with 5 mcg/ml IgM. (B) B cells were purified from Pacs1+/+ and Pacs1-/- spleens and OCR was measured using a Seahorse metabolic flux analyzer. OCR after rotenone/Antimycin A treatment was subtracted from baseline OCR to give basal mitochondrial respiratory rate. Data shown are the mean of 7-10 technical replicates from three independent experiments. Error bars indicate mean ± SD. Two-tailed unpaired t test, *P < 0.05. (C) Determination of cellular ROS in Pacs1+/+ and Pacs1-/- FOB cells with CellRox Green. Quantification shows the mean fluorescence intensity (MFI) from three separate pairs of Pacs1+/+ and Pacs1−/− mice. Two-tailed paired t test, **P < 0.01. (D) Splenocytes from Pacs1+/+ and Pacs1−/− mice were stained with cell surface antibodies to identify FOB cells and treated with 100 µM H2O2 for 35 minutes. Cells were then labelled with TMRE to monitor MMP by FACS analysis. Low TMRE fluorescence indicated susceptibility to H2O2 treatment. Data are presented as mean ± SD of 3-4 replicates, two-tailed unpaired t test, ***P < 0.001. Results are representative of three independent experiments performed on different Pacs1+/+ and Pacs1−/− pairs.
Fig.Figure 5A - H Genetic interactions between PP2A-29B and actin regulators during dendrite pruning. The indicated manipulations of actin regulators were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. (A) C4da neuron coexpressing PP2A-29B dsRNA and tdtomato as suppression control. (B) C4da neuron coexpressing PP2A-29B dsRNA and GFP::tsr. (C) C4da neuron coexpressing PP2A-29B dsRNA and RacN17. (D) C4da neuron coexpressing PP2A-29B dsRNA and RhoN19. (E) C4da neuron expressing PP2A-29B dsRNA in ena210/+ background. (F) C4da neuron coexpressing PP2A-29B dsRNA and Orco dsRNA as suppression control. (G) C4da neuron coexpressing PP2A-29B dsRNA and Chicadee dsRNA. (H) C4da neuron coexpressing PP2A-29B dsRNA and moesin dsRNA. I Penetrance of pruning defects in panels E - L. n. s., not significant, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure. J Severity of pruning defects in panels E - L as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, ** P<0.005, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.
Generation of a local knockdown of α1S subunit.Figure 1(A) Schema of exonic chaining of pre‐mRNA, exons 15, 16 and 17 are represented by blue boxes. Sequences of exon acceptor site SA (in blue) and exon splicing enhancer ESE (in black) of exon 16 were targeted by antisense sequences introduced in U7smOPT cassettes. (B) qRT-PCR analysis using primers overlapping exons 15 and 16 permitted to quantify the non‐skipped α1S form: 18±6% (**P⩽0.001, n=19) and 10±7% (**P⩽0.001 n=19) (black bars) of the total α1S mRNA (grey bars) at 2 and 6 months post injection in TA of AAV1‐(U7‐ESE) and AAV1‐(U7‐SA):ΔDHPR or AAV1‐(U7‐Ctrl): c as a control, respectively. (C) Six months post injection, lysates from TAΔDHPR (ΔDHPR) and TACtrl (c) were immuno‐blotted for α1S or α-actin for four mice. Graph depicts mean±s.e.m. of relative expression of α1S subunit determined by densitrometry and nomalized to the α-actin expression for each muscle. Results were expressed in protein levels of α1S subunit in TAΔDHPR normalized to TACtrl for each mice, **P⩽0.001, n=4. (D) Longitudinal cryo‐sections from TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐α1S subunit (red) and anti‐laminin (green) antibodies, nuclei were visualized by Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm.
Contribution of lab-specific factors to the reproducibility of functional gene dependency profiles and MS-based proteomic profiles.Figure 2(A) Correlation matrix plot of average Spearman correlation of gene dependency profiles of cancer cell lines calculated based on genome-wide RNAi screens and CRISPR screens. Number of overlapping cell lines between any two datasets used for estimating the average correlation ranges between 2 - 284, with a mean of 46.4. The empty cells indicate that no identical cell lines were profiled between the two datasets. (B) Distribution of Spearman correlation of gene dependency profiles between different study sites. Triangles represent mean correlation values. Numbers below the labels represent the number of overlapping cell lines based on which the distributions were drawn. (C) Average Spearman correlation of MS-based proteomic profiles between different study sites generated using different peptide labeling procedures. The empty cells indicate that no identical cell lines were profiled between the two datasets. Number of overlapping cell lines between any two datasets used for estimating the average correlation ranges between 3 - 27, with a mean of 7.8. (D) Distribution of Spearman correlation of MS-based proteomic profiles between different study sites. Numbers below the labels represent the number of overlapping cell lines based on which the distributions were drawn. Triangles correspond to the median value. (E) Coefficient of variation (CV) of proteins detected and quantified in UW TNBC study (non-TMT labeled) vs. MGHCC BREAST study (TMT-labeled). Both studies had a maximal overlap of breast cancer cell lines for a robust estimation of CV. Housekeeping genes are highlighted as red dots. Spearman correlation (rcv) was calculated to estimate the agreement in the CV estimates of common set of proteins between the two studies.
Figure 1A. Single grey-scale and coloured merged images of immunocytochemical staining against PSD-95, synaptophysin-1 (SYP1) and NPC1 proteins in a cultured hippocampal neuron from a wt mouse. Graphs show mean ± SEM of percentage of colocalization of NPC1 with PSD95 or SYP1 (n=12 neurons from 3 different cultures). B. Electron microscopy image of immunogold labelling against NPC1 in the CA1 hippocampal region of a wt mouse. Presynaptic compartments are indicated in pink while postsynaptic in blue. White arrows show the 15-nm gold particles linked to NPC1 (d-dendrite, sv-synaptic vesicles, m-mitochondria). C, D. Western blots against NPC1 and actin-β (ACTB) in total brain (C) and synaptosomes (D) from wt and NPC1nmf164 mice. Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control (wt) values (n=4 mice, 3 month-old, unpaired Student t test, **psynaptosomes= 0.0037). E. Western blots against NPC1 and actin-β (ACTB) in total brain extracts from wt and Npc1-null mice (NPC1KO) containing the same amount of protein (n=2 mice, 6 week-old). F. Graph shows mean ± SEM mRNA levels of NPC1 in total extracts and in synaptosomes from wt and NPC1nmf164 mice (n=4 mice, 3 month-old). G. Western blots against NPC1 and actin-β (ACTB) in synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not in the presence or absence of the protein synthesis inhibitor cycloheximide (CHX). Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control (wt non cLTP induced) values (n=3 mice, 3 month-old, 2-way ANOVA, ***p < 0.001). H. To the left Western blots against NPC1 and actin-β (ACTB) in synaptosomal extracts from wt and NPC1nmf164 mice used as input for the immunoprecipitation assays shown in the right. Immunoprecipitates were pulled down with an anti-ubiquitin antibody and without it for negative control and analysed by western blot against NPC1. MG132 was used as proteasome inhibitor. Graphs show mean ± SEM levels of NPC1 normalized to ACTB in the inputs (left) (n=3, one-way ANOVA, *pWT+MG-132=0.0496, pNPC1nmf1+MG-132=0.0358) and of NPC1 associated to ubiquitin (right) (n=3 mice, 3 month-old, one-way ANOVA, *pNPC1nmf164= 0.0237, pNPC1nmf164+MG-132= 0.0386.
Figure 1(a, b) Dimension reduced single cell transcriptomic data is visualized through Uniform Manifold Approximation and Projection (UMAP). The color code illustrates the disease status (a) and cell type identity (b) (c-f) The indicated marker genes were used to select clusters for subsetting into stromal cells (c), epithelial cells (d), endothelial cells (e), and leukocytes (f). (g-j) The heatmaps show the relative gene expression levels for the indicated marker genes for the indicated stromal (g), epithelial (h), endothelial (i), and leukocyte (j) cell types.
Spontaneous proliferation and increased cell death of Pacs1−/− B cells in vivo under lymphocyte replete conditions (A) Pacs1+/+ and Pacs1−/− B cells were purified, labeled with CTV dye, and stimulated with the indicated mitogens.Figure 7(A) Pacs1+/+ and Pacs1−/− B cells were purified, labeled with CTV dye, and stimulated with the indicated mitogens. Cell proliferation was assessed after 72 h with FACS analysis based on CTV dilution. (B, C) Pacs1+/+ and Pacs1−/− mice were immunized with alum-ova and one week later with NP-Ficoll. Anti-ova IgG and anti-NP IgM titers were measured at 14 days and 7 days after immunization, respectively. Each symbol represents an individual mouse. (D) Pacs1+/+ and Pacs1ccy/ccy mice were immunized with NP-KLH. Low affinity (anti-NP30) and high affinity (anti-NP2) antibodies were measured 14 days after immunization. (E) B cells were purified from Pacs1+/+ and Pacs1−/− mice and labeled with CTFR and CTV dyes, respectively. Labeled B cells were injected into unirradiated CD45.1 recipients at ~1:1 ratio. Proliferation and survival of adoptively transferred B cells were measured 8 days post-transplant. (F) Fraction of donor B cells that proliferated after adoptive transfer from independent experiments using three different Pacs1+/+ and Pacs1−/− donor pairs. Each symbol represents an individual recipient. Horizontal bars indicate median value. Two-tailed unpaired t test, **P < 0.01, ***P < 0.001. (G) Fraction of donor B cells that were Annexin V positive after adoptive transfer from two independent experiments using two different Pacs1+/+ and Pacs1−/− donor pairs. Each symbol represents an individual recipient. Horizontal bars indicate median value. Two-tailed unpaired t test, **P < 0.01, ***P < 0.001. (H, I) Pacs1+/+ and Pacs1-/- mice were injected with EdU and the fraction of EdU+ FOB and MZB cells were measured in the spleen at 1, 4, and 7 days post-injection. Data are from one experiment.
Protocadherin‐7 as a mitotic markerWorkflow of flow cytometry analysis.Figure 6A Workflow of flow cytometry analysis. B Flow cytometry analysis of HeLa cells treated with anti‐PCDH7 antibody, fixed and stained for phospho‐Histone H3 and DNA. Forty‐two percent of unsorted HeLa cella are in G2/M phase. When cells are sorted based on their phospho‐Histone H3 (middle) expression, phospho‐Histone H3+ (high expression) cells are preferentially mitotic (87.3% in G2/M phase) and 42% of those are PCDH7+. Similarly, PCDH7+ (high expression) cells are preferentially mitotic (82.1% in G2/M phase) and 79% of those are phospho‐Histone H3+ (bottom).
Figure 1Determination of serum testosterone (B) and 17β-estradiol levels, in post-pubescent male, castrated male (CtM), and female Hanwoo (n = 5 in each group). Determination of the body weight , in post-pubescent male, castrated male (CtM), and female Hanwoo (n = 5 in each group). D. PCoA of the rectal bacterial 16S rRNA gene sequences based on the weighted UniFrac distance matrix. E. Discriminant taxa of the male and CtM rectal microbiota, as determined by the linear discriminant analysis effect size (LEfSe). The different abundances are represented by LDA scores. F. The serum metabolome profiles were analyzed using GC-TOF-MS and clustered by PCoA based on the Bray-Curtis dissimilarity matrix. G. The relative abundances of serum metabolites displayed as bar graphs (n = 5 in each group). H. The BCAA serum levels quantified enzymatically, as described in the materials and methods section.
Figure 2(A) L2 was tagged with HA at the C-terminus and expressed from the doxycycline-inducible promoter. (B) Western blotting of WT MEFs, Drp1-KO MEFs, and Drp1-KO MEFs carrying Drp1, all of which express L2-HA, using the indicated antibodies. The expression of L2-HA was induced for 16 h (0.1 µg/ml doxycycline). The asterisk indicates non-specific bands of anti-Oma1 antibodies. (C) Quantification of band intensity. Values are average ± SD (n = 3). (D) The activation of Oma1. Oma1 is proteolytically activated and then undergoes degradation. (E) WT and Drp1-KO MEFs, both of which express L2-HA, were transduced with lentiviruses carrying either scramble or Oma1-targeted shRNAs. Whole-cell lysates were analyzed by Western blotting using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (F) Quantification of band intensity. Values are average ± SD (n = 3).
Figure 2Figure 2. Dynamic reorganization of chromosomes during replication.(A) FACS profiles and representative DAPI-stained cells of G1 synchronized cells released in S phase. Figure 2. Dynamic reorganization of chromosomes during replication. (B) PCA analysis of the distance matrix between the contact maps of the population displayed in (A). Figure 2. Dynamic reorganization of chromosomes during replication. (C) p(s), i.e., average intra-chromosomal contact frequency p between two loci with respect to their genomic distance s along the chromosome (log-log scale) during replication (color code identical to FACS profiles and PCA). Figure 2. Dynamic reorganization of chromosomes during replication. (D) Left panel: p(s) of replication defective cells (cdc6 thermosensitive mutant). G1 elutriated cells were released for 3h and 6h in non-permissive conditions. The corresponding FACS profiles show no S-phase progression. Middle panel: p(s) of cells that completed replication. G1 elutriated cells were released in S phase in the absence or presence of nocodazole (G2/M noc). Right panel: p(s) of cohesin depleted (ssc1 G2) and nocodazole arrested cells. Figure 2. Dynamic reorganization of chromosomes during replication. (E) Log-ratio of contact maps between G2 and G1 cells (top right) and ssc1 G2 and G1 cells (bottom left). Blue to red color scales reflect the enrichment in contacts in one population with respect to the other (log2). Yellow arrowheads: inter-centromere contacts. Figure 2. Dynamic reorganization of chromosomes during replication.. (F) Normalized contact frequencies between chr3 centromere (cen3; yellow arrowhead) and the rest of the genome for G1, G2 and ssc1 G2.
Apg16p mediates Apg5p assembly.Figure 6Apg16p mediates Apg5p assembly. Immunoprecipitation was carried out as described in Figure 3, using apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115) carrying pMyc‐APG12 (CEN) and pHA‐APG12 (CEN) (A), or pMyc‐APG12 (CEN) and pHA‐APG5 (CEN) (B). Following SDS-PAGE, immunoprecipitates were subjected to immunoblot analysis using anti‐HA or anti‐Myc antibody as indicated. A model of the Apg12p-Apg5p-Apg16p complex is illustrated in each panel. Apg16p forms a homo‐oligomer through its C‐terminal coiled‐coil region. It is not clear whether Apg16p forms a dimer or a larger multimer. The N‐terminal region of each Apg16p interacts with Apg5p; most of these complexes are conjugated to Apg12p (A) but a small population are unmodified (B). In the absence of Apg16p, separate Apg5ps do not assemble. Apg16p mediates Apg5p assembly. Immunoprecipitation was carried out as described in Figure 3, using apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115) carrying pMyc‐APG12 (CEN) and pHA‐APG5 (CEN) (B). Following SDS-PAGE, immunoprecipitates were subjected to immunoblot analysis using anti‐HA or anti‐Myc antibody as indicated. A model of the Apg12p-Apg5p-Apg16p complex is illustrated in each panel. Apg16p forms a homo‐oligomer through its C‐terminal coiled‐coil region. It is not clear whether Apg16p forms a dimer or a larger multimer. The N‐terminal region of each Apg16p interacts with Apg5p; most of these complexes are conjugated to Apg12p (A) but a small population are unmodified (B). In the absence of Apg16p, separate Apg5ps do not assemble.
Figure 3Cartoon representation of the 1.5 Å W. pipientis wMelOTU crystal structure with labeled termini, missing regions, and features of the active site. Structural alignment of the core OTU folds (central β-sheet and two supporting α-helices) from human OTUB1 (green, PDB 4DDG) and wMelOTU (purple). Surrounding regions are less-well conserved and shown as semi-transparent. Enlarged region of the OTUB1:Ub structure (PDB 4DDG) showing entry of the Ub C-terminus (red) into the OTUB1 active site (green). The wMelOTU structure (purple) is overlaid to highlight the structural conflict between the downward position of the His-loop and the Ub C-terminus. 1.8 Å crystal structure of the covalent wMelOTU-Ub complex. wMelOTU (cartoon, pink) is linked to the Ub (surface red) C-terminus through its active site. Primary, secondary, and tertiary regions of the Ub-binding S1 site are indicated. Structural overlay of the apo (violet) and Ub-bound (pink) wMelOTU structures highlighting the repositioning of the His-loop to accommodate entry of the Ub C-terminus, as well as ordering of two regions in the S1 site that form an embrace around Ub. Detailed view of the primary and secondary interfaces between wMelOTU (pink) and Ub (red) observed in the wMelOTU-Ub structure. wMelOTU and Ub residues participating in the interface are shown with ball and stick representation. Ub-KG(TAMRA) cleavage assay monitoring the effects of structure-guided wMelOTU mutations. These data were collected in parallel with those presented in Fig. 1G and the WT dataset is shown again for reference. Detailed view of the wMelOTU (pink) active site region and its coordination of the Ub C-terminus (red). Residues that coordinate Ub or stabilize the active site are shown with ball and stick representation.
Cancer-secreted EVs suppress AA-induced mTORC1 signaling and protein synthesis in fibroblasts.Figure 1A. CAF and WI-38 cells pretreated with EVs from MDA-MB-231 or MCF10A cells, or PBS for 48 h were unstarved (U) or starved (S) for 2 h in EBSS prior to addition of a mixture of leucine, arginine, and lysine (LRK) or a cocktail of essential AAs (EAA). Cells were analyzed by Western blot analysis in a time course as indicated. B. Western blot analysis of puromycin (Puro)-labeled fibroblasts (CAF, normal human mammary fibroblasts, WI-38, and NIH3T3) after 2-h starvation and EAA add-back in the presence or absence of cycloheximide (CHX). C. Western blot analysis of Puro-labeled fibroblasts cultured in growth medium.
Figure 6(A) Western blotting of WT MEFs and Drp1-KO MEFs, both of which carry shRNAs (Oma1 or scramble) and/or ectopic Mfn1. The asterisk indicates non-specific bands of anti-Oma1 antibodies.(C-F) WT MEFs (C and E) and Drp1-KO MEFs (D and F) were transduced with Su9-Eos, along with Oma1 shRNAs and ectopic Mfn1. Su9-Eos in the small region of mitochondria (indicated by the asterisks) was photoconverted using a 405-nm laser on confocal microscope. Within 1 s after photoconversion, images were obtained for both photoconverted and unconverted Su9-Eos signals. Images before the photoconversion were also taken to detect background signals. Scale bar, 10 µm. (E and F) To determine the connectivity of mitochondria, the area containing photoconverted Su9-Eos signals was divided by the area containing the photoconverted and unconverted Su9-Eos signals (total mitochondria). Values are average ± SD (n = 30 cells). (G) The number of mitochondria was estimated by reversing the connectivity. (H) Summary of the data.
UBE4B and LSD1 knockdown activates transcription mediated by p53.Fig 4(A) Venn diagram of upstream activators (z-score ≥ 2) that are differentially activated in single-UBE4B or LSD1-knockdowns and double UBE4B and LSD1 knockdowns, compared with the control. Activation state of an upstream regulator is predicted from differential mRNA levels of its downstream target genes. (B) The volcano scatter plot indicates fold changes in the levels of gene transcripts affected differentially by the UBE4B and LSD1 double knockdown versus control shRNA. Gray spots represent 22,148 annotated transcripts. Red spots are predicted p53-activated targets, and blue spots are predicted p53-inhibited targets. The enrichment of red spots in the up-regulated genes (right upper quadrant, >1.2-fold change, p ≤ 0.04) and blue spots in the down-regulated genes (left upper quadrant) indicates that the p53-mediated transcription is activated by the UBE4B and LSD1 double knockdown. C) RT-qPCR validation of the expression levels of representative p53 target genes in the UBE4B and LSD1 double-knockdown samples. n = (2 to 6), p ≤ 0.04. (D) The p53 protein level is significantly increased by UBE4B and LSD1 double knockdown. Left: representative western blots showing p53 levels, the knockdown of UBE4B and LSD1, and the actin control in HEK293T cells. Right: quantification of the p53 protein levels normalized against actin (n = 3). E) The p53-mediated transcriptional activity is measured by a luciferase reporter under the control of a p53-response element promoter, which was transfected into HEK293T cells 72-96 h after the initiation of the UBE4B and LSD1 single or double knockdowns (n = 6). (F) Increased interaction of p53 with its activating partner 53BP1 in response to UBE4B and LSD1 knockdown. Left: representative western blots of 53BP1 co-immunoprecipitation. Right: quantification of the 53BP1 co-immunoprecipitation (n = 2). Data represent means ± SEM. (G) RT-qPCR validation of the expression levels of FOXO3a, FOXO4, and PSMD11 (n = 6). (H) The FOXO3a-mediated transcriptional activity was measured with a luciferase reporter under the control of a FOXO-response element promoter. HEK293T cells were transfected with shRNAs for control, LSD1, UBE4B, or both LSD1 and UBE4B, followed by transfection of the luciferase reporter together with a constitutively active form of FOXO3a (TM) 72-96 h later (n = 6). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Opsonized AdV- dependent IL-1β and TNF release can occur without concurrent cell death.Figure 2AdV-GFP was incubated with 20 ug/ml h9C12 antibody or 20 mg/ml IVIg in PBS. These AdV-Ab complexes were then diluted 1:2 to give final doses of viral particles being added to WT THP-1 cells (with antibody complexed) as indicated. A single experiment representative of 3 independent experiments is shown. THP-1s were stimulated with a constant dose of AdV-GFP (50 000 pp/cell), and complexed with decreasing antibody concentrations as indicated. A single experiment representative of 2 independent experiments is shown. HMDM were stimulated directly (TNF) or primed (IL-1β, LDH) with AdV-Ab complexes as in (B). Data for each individual donor (2 total) is shown. THP-1 were stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell death was measured by LDH release THP-1 were stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell viability measured by PrestoBlue assay HMDM were primed, or not, and stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell death was measured by LDH release HMDM were primed, or not, and stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell viability measured by PrestoBlue assay