EMBO/sd-character-level-ner huggingface dataset:
Figure 4Comparison of gene expression in Mta123∆ ES cells and wild type (WT) ES cells. Each circle represents a gene: red indicates spike-in controls, blue indicates genes that are not differentially expressed to a significant degree, and green indicates differentially expressed genes (2404 increased, 1293 decreased) defined with an adjusted p-value < 0.05 and a log2 fold-change > 1. N=3 for each genotype. Fold change in gene expression is plotted for different subsets of genes. All genes are plotted in black, while subsets of genes located nearest to ChIP-seq peaks for the indicated proteins +Chd4 and which show significant changes in expression compared to wild type cells are plotted in dashed coloured lines as indicated. The number of genes in each of the Mta categories are: all genes (32271), all differentially expressed (n=3701), Mta1+2+3 (n=1738), Mta1+2 (n=1460), Mta1 (n=1020) and Mta2 (n=924). Principal component analysis of RNAseq data from ES cell lines of indicated genotypes in either self-renewing conditions (2i) or after 48 hours in the absence of two inhibitors and LIF (Diff48). Each point represents a biological replicate. GO term enrichment for genes differentially expressed in Mta123∆ ES cells (left) or Mbd3∆ ES cells (right) compared to wild type cells in 2iL conditions. For each comparison the top five most significant gene ontology terms are plotted by log10 of the Benjamini-adjusted p-value. The significant GO terms and p-values were calculated using David v.6.8 Genes associated with indicated GO terms plotted by fold change in expression in Mta123∆ ES cells (x-axis) or Mbd3∆ ES cells (y-axis). Genes are coloured if they are differentially expressed (log2 fold-change > 1 and adjusted p-value < 0.05) in either comparison as indicated. The dotted lines show the fold-change cut-off of 2. GO-terms were identified using David v.6.8 using a Benjamini score with a cutoff of 0.05. Genes associated with core NuRD peaks, with an MTA-only peak, with a Chd4 peak and an MTA-peak but not an Mbd3 peak (Chd4+Mta-Mbd3 genes), or with an MTA-peak but not a Chd4 peak (MTA-only genes) were divided into quartiles based upon expression levels in wild type cells. Change in gene expression is plotted on the x-axis and calculated significance of the expression change on the y-axis. Those genes significantly misexpressed (|log2FC|>1; p≤0.05) are indicated in red, genes showing no significant change are plotted in black. The percentage of genes within each quartile which is significantly misexpressed is indicated at the top of each plot.
Inhibiting nuclear import leads to smaller nuclei and cytoplasmic lamin aggregates.Figure 5MCF-10AT1k.cl2 cells were transfected with plasmids expressing mCherry alone or mCherry-RanQ69L and stained with an antibody against lamin B2. Representative images are shown. MCF-10AT1k.cl2 cells were treated with 20 µM importazole or an equivalent volume of DMSO as a control for 24 hours and stained with an antibody against lamin B1. Representative images are shown. For each experiment, nuclear cross-sectional areas were quantified for 32-271 nuclei per condition (123 nuclei on average) and averaged. Three biological replicates, data from one representative experiment shown. To measure bulk import capacity for importin α/β cargos, experiments were performed as in (A-B) except that cells were also transfected with a plasmid expressing GFP-3x SV40 NLS. For each experiment, nuclear GFP-NLS intensities were quantified for 11-75 nuclei per condition (35 nuclei on average) and normalized to the appropriate control. Two biological replicates, data from one representative experiment shown. Cells with and without cytoplasmic lamin B aggregates were counted and the percentage of cells with aggregates was calculated. For each experiment, 31-130 cells were examined per condition. Three biological replicates, data from one representative experiment shown.
Figure 5Evaluation of lung micrometastases using HE and vimentin-stained lung sections upon antiangiogenic therapies in Ren13BM Representative images of lung metastasis in Ren13BM upon antiangiogenic therapies. (B) Incidence (pie chart, *p < 0.05 by Chi-square) and proportion of mice with 0, 1, 2 or > 3 metastases (mets) per lungs Evaluation of lung micrometastases using HE and vimentin-stained lung sections upon antiangiogenic therapies in Ren13BM and Ren28. proportion of mice with 0, 1, 2 or > 3 metastases (mets) per lungs (*p < 0.05 by Mann-Whitney test) are shown.Anti-VEGF/R treatments exacerbate the invasive capacity quantified as capsular invasion in the tumor kidney interface in some Ren-PDOXs. Moreover, the evaluation of lung metastasis confirms the pro-malignant effects of antiangiogenics in some Ren-PDOXs, recapitulating the inter-patient variable response.
Figure 8In healthy cells Stx17 facilitates normal division of mitochondria (Mt) by interacting with Drp1. Upon mitophagy stimulation, Stx17 dissociates from Drp1 and interacts with Atg14L for autophagosome formation. In the case of autophagy, mitochondria elongate due to inactivation of Drp1, whereas in mitophagy, FUNDC1 (FDC1) is dephosphorylated by cleaved PGAM5 (PGAM5*), which facilitates excessive division of mitochondria (Mt*) by interacting with Drp1. Stx17 supports this process by releasing PGAM5. Under normal division conditions, FUNDC1 associates with CNX through unknown protein(s).
Figure 7(A) Differential interference contrast images (left panels) and confocal microscopy analysis of IL-18 (right panels) in a DC/NK cell conjugate after 2 hours of coculture in the presence of LPS and in unstimulated cells (NT). Representative images from 3 experiments are shown, magnification 63X. Scale bars, 4.36 m. (B) IFN-γ release by NK cells cultured in the presence of supernatants recovered from untreated DCs (untreated) or DCs treated O/N with LPS (LPS). Where indicated IL-18 was added to the cultures. (C) Cell-contact dependent activation of NK cells by DCs depends on IL-18. Unstimulated or LPS-activated DCs and NK cells were cocultured in the same wells (DC + NK + LPS) or separated by a porous membrane ((DC+LPS)/NK). Where indicated IL-18 was added to the transwells ((DCs+LPS)/(NK+IL-18)). NK cells alone were also cultured in the presence of the three selected cytokines; IL-2 and IFN-β in the upper chamber and NK cells and IL-18 in the lower chamber. IFN-γ in the supernatant was then measured by ELISA after 8 hours of coculture. (B, C). n (number of independent experiments)=3. Error bars depict SEM.
Figure 6A. Mitochondria isolated from tumors (T) or adjacent normal (N) tissues of WT and OMA1-/- mice were subjected to blue native PAGE (BN-PAGE), and respiratory chain complexes were analyzed by Western blotting with anti-NDUFB8 (Complex I), anti-SDHA (Complex II), anti-COX4L1 (Complex IV), and anti-ATP5A1 (Complex V, ATP synthase) antibodies. SDS-PAGE with antibody against HSPD1 was used as a loading control. Relative protein levels were further evaluated by densitometry analysis using ImageJ software. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001. B. Digitonin-solubilized mitochondria were isolated from control and OMA1 KO HCT116 cells cultured in normoxia or hypoxia (1% O2) for 12 hours, and then analyzed by BN-PAGE and immunoblotting using the indicated antibodies against NDUFB8 (Complex I), SDHA (Complex II), NDUFA4 (Complex IV), and ATP5A1 (Complex V). SDS-PAGE with antibody against HSPD1 was used as a loading control. Relative protein levels were further evaluated by densitometry analysis using ImageJ software. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). N.S., not significant, *p<0.05. C. The lysates of adjacent normal tissues (N) or tumors (T) from WT and OMA1-/- mice were analyzed by Western blotting with antibodies against NDUFA4, COX4L1, NDUFB5, DNUFB6, UQCRC2, or Tubulin. Relative protein levels were further evaluated using ImageJ software. Error bars indicate the mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, ***p < 0.001. D. A graphic model of mitochondrial stress sensor OMA1-coordinated glycolysis and mitochondrial oxidative phosphorylation in colorectal carcinogenesis and development. Hypoxia-induced OMA1 activation leads to the degradation of mitochondrial respiratory chain complexes (MRCC) components and OPA1, and results in cristae remodeling, all of which results in the inhibition of mitochondrial electron transport chain (ETC) and the generation of mitochondrial ROS. Elevated ROS can stabilize HIF1a, thereby promoting glycolysis. Thus, OMA1 promotes the Warburg effect by coordinating glycolysis and oxidative phosphorylation to facilitate colorectal cancer development.
Figure 5A Lysates of 293T cells transfected with plasmid for Flag-Beclin-1 and HA-K11-linked-Ub, together with empty vector or expression vector for Myc-USP5 or Myc-USP19 were immunoprecipitated with anti-Flag and immunoblotted with anti-HA. B Immunoprecipitation and immunoblot analysis of 293T cells transfected with vectors expressing Flag-Beclin-1 and HA-K11-linked ubiquitin. C 293T cells were transfected with Flag-Beclin-1 (WT or K437R) and treated with CHX (100 μg/ml) for indicated time points. The expression levels of WT and K437R Flag-Beclin-1 were analyzed by immunoblot.D Quantification of the expression levels of WT and K437R Flag-Beclin-1. E Immunoblot analysis of protein extracts of 293T cells transfected with empty vector or vector for HA-USP19, together with plasmid expressing WT or K427R, K430R or K437R mutant of Flag-Beclin-1. F HeLa cells transfected with BECN1 sgRNA and restored with WT and K437R Flag-Beclin-1 were stained by anti-LC3 antibody. Scale bar, 200 μm.G Quantification of LC3 puncta shown in (F). Bars represented mean ± SEM of triplicate samples. *p < 0.05, **p < 0.01 and ***p < 0.001.
Figure 3A) Outline of the SATAY screening procedure. Cells expressing plasmids containing the PE and PC synthesizing enzymes and a plasmid harboring the galactose inducible transposase (TPase) and a transposon (TN) disrupting the TRP1 gene were grown for ~56h in galactose-containing medium to induce transposition (step 1). Cells were inoculated in synthetic medium lacking tryptophan in either KennedyON or OFF conditions, grown for several generations (step 2) and harvested for DNA extraction and sequencing of transposon insertion sites (TNs) (step 3). TNs are mapped to the genome to identify genes that become required under certain rewired conditions (step 4). An illustrative example of TN insertions of four libraries visualized in the UCSC genome browser highlights a genetic requirement for DRS2 (green bar) in KennedyOFF conditions. B) GO term enrichments in the top 200 (red) and top 500 (blue) most variable genes (defined as genes with the highest standard deviation of TN insertions across libraries) identified using the YeastMine web server. Significance was determined using Holm-Bonferroni corrected p-values with a cut-off of 0.05, the size of the circles is inversely proportional to the p-values (i.e. bigger circles indicate higher significance). C) VPS13 is required in KennedyOFF conditions when chimeric enzymes are targeted to mitochondria or endosomes. Volcano plots show the fold change of number of transposon insertions per gene of libraries grown in KennedyOFF versus ON conditions. This comparison included all six libraries where the enzymes are targeted to endosomes and/or mitochondria (top left panel) or six libraries with enzymes targeted to other organelles (bottom left panel). Data-points for VPS13 are highlighted in red. Schematic illustrating Vps13-mediated lipid transport at endosomes and mitochondria contact sites (right panel). D) Schematic (top panel) illustrating the requirement for SAM transport across the mitochondrial inner membrane by Sam5 for PC production by Pmt in the mitochondrial matrix (MM). Lipids can be produced by the Kennedy pathway (red) independent of Sam5 activity. Transposon insertion maps generated in the UCSC genome browser for SAM5 for the library where PC is produced in the MM in either KennedyON or OFF conditions (bottom panel).
Figure 7(A) Recombinant Flag-GP130, Myc-NEDD4L, Myc-NEDD4L-ΔC2, Myc-NEDD4L-ΔWW, Myc-NEDD4L-ΔHECT or Myc-NEDD4L-C943S, and ubiquitination-associated proteins were mixed in adenosine 5'-triphoshpate buffer and incubated at 30℃ for 2 h. Then the reaction aliquots were quenched and measured by WB with anti-Flag antibodies. (B) Whole cell lysates of WT and NEDD4L KO NHEKs were immunoprecipitated with anti-GP130 antibody, and the immune-precipitates were analyzed by WB with anti-Ub antibodies. (C) WT or NEDD4L KO NHEK cells was infected with lentivirus that overexpress NEDD4L-WT, NEDD4L WW domain truncated mutant (NEDD4L-ΔWW) or its enzymatic activity mutant (NEDD4L-C943S), the cells were followed by denaturation-IP with anti-GP130 antibody and WB analysis with anti-Ub antibodies. (D) WB analysis of the polyubiquitination of GP130 in HEK293T cells co-transfected with Flag-GP130, Myc-NEDD4L, HA-Ub or its mutants expressing only one lysine (K-only), respectively, which followed by denaturation-IP with ANTI-FLAG M2 beads and WB analysis with anti-HA antibodies. (E) WB analysis of the polyubiquitination of GP130 in HEK293T cells co-transfected with full length Myc-NEDD4L, HA-Ub and Flag-GP130 or its lysine mutants (K-R) in the intracellular domain, followed by denaturation-IP with ANTI-FLAG M2 magnetic beads and WB analysis with anti-HA antibodies.
Figure 1(A) GFP-PAQR3 transfected HeLa cells were fixed for immunofluorescence staining with indicated antibodies before or after glucose starvation (GS) for 4 h. The nuclei were stained with Hoechst 33342. Scale bar: 10 μm. (B) Immunoblotting (IB) analysis of wild type (WT) and PAQR3-deficient MEFs after GS for different time as indicated. (C) WT and PAQR3-deficient MEFs were treated with 80 mM chloroquine (CQ) for 2 h to induce LC3-II accumulation. Whole-cell lysates were harvested for IB analysis of LC3-II. (D) Autophagosome-like structures in WT and PAQR3-deleted MEFs were detected after GS for 4 h by transmission electron microscopy. * indicates autophagosome-like structures. N, nucleus.(E) Quantitative analysis of autophagic vacuoles in transmission electron microscopy images. Thirty cells were quantified from each independent experiment，which was repeated for three times with similar results. Values are presented as mean ± SD, ***p < 0.001. (F) Immunofluorescence staining of LC3 in WT or PAQR3-deficient MEFs after GS for 4 h. The arrowhead indicates representative LC3-positive puncta. Scale bar: 10 μm. (G, H) HeLa cells were infected by PAQR3 knockdown or overexpression lentivirus together with their respective control virus. After GS for 1 h, the cell lysates were harvested for IB.
Figure 7(A) In vitro kinase assays with the indicated recombinant protein in the presence of [γ-32P]-ATP for 30 min. Phosphorylation was detected by autoradiography and levels of proteins were visualized by coomassie blue staining. Data information: Scale bar: 5 μm. (B) ClustalW sequence alignment for Spindly orthologues. The region encompasses the Polo-dependent phosphorylation site identified in Drosophila Spindly (red box). Asterisk highlights a conserved residue identified in silico by the Eukaryotic Linear Motif (ELM) resource as a putative Polo-dependent phosphorylation site in human Spindly. Sequence alignment was coloured according to clustal colour scheme in Jalview (www.jalview.org, v2.10.5). Abbreviations: h, Homo sapiens; m, Mus musculus; x, Xenopus laevis; d, Drosophila melanogaster. Data information: Scale bar: 5 μm. (C) Sequence alignment showing that the Polo-dependent phosphorylation site in Drosophila Spindly lies within a conserved region found in different dynein adaptors. Abbreviations: h, Homo sapiens; m, Mus musculus; d, Drosophila melanogaster. Sequence alignment was coloured according to clustal colour scheme in Jalview (www.jalview.org, v2.10.5). The binomial log10 sequence logo was created using the PSSMSearch software (Krystkowiak et al, 2018). Data information: Scale bar: 5 μm. (D) Representative immunofluorescence images of Spindly-phospho(ph)Ser499 levels at unattached KTs in control Drosophila S2 cells and in Polo-depleted or BI2536-treated cells. Insets display magnifications of the outlined regions. Cells were treated with colchicine prior to fixation to generate unattached KTs. CID was used as a KT reference. Data information: Scale bar: 5 μm. (E) Graph represents the levels of Spindly phS499 at unattached KTs for cells Spindly phS499 levels were determined relative to CID and all values were normalized to the control mean fluorescence intensity, which was set to 100% (n≥1246 KTs from at least 63 cells for each condition, n=2 independent experiments). Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ****, <0.0001 Data information: Data are shown as mean ± SD. Scale bar: 5 μm. (F) Representative immunofluorescence images of Spindly phS499 levels at unaligned and aligned KTs in a PoloWT-EGFP expressing Drosophila S2 cell. Insets show magnifications of the outlined regions, which highlight either a unaligned (u) KT or an aligned (a) KT. CID was used as a KT reference. Plotted profiles of signal intensities of phS499 and CID are shown for the highlighted KTs. Data information: Data are shown as mean ± SD. Scale bar: 5 μm. (G) Graph represents the levels of Spindly phS499 at unaligned KTs versus aligned KTs for cells Spindly phS499 levels were determined relative to CID and all values were normalized to the mean fluorescence intensity of unaligned KTs, which was set to 100% (n≥34 KTs from 14 cells, n=2 experiments). Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: ****, <0.0001. Data information: Data are shown as mean ± SD. Scale bar: 5 μm.
Tamoxifen reduces LOX-L2 levels in PSCs and PDAC tissues.Figure 2(A) qPCR levels of LOX-L2 in PSCs, normalized to RPLP0 and relative to control (B) Western blot levels of LOX-L2 in PSCs (n=3 experimental replicates, **P<0.01) (C) Expression of LOX family genes obtained from RNA-seq data in control and tamoxifen treated PSCs (n = 3 experimental replicates). Expression value was normalized by tubulin family genes (D) qPCR levels of LOX family in PSCs, normalized to RPLP0 and relative to control (E) Relative values of protein levels for LOX members in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates (F) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxifen, scale bar 50 μm. (G) Quantification of LOX-L2 for images in F. n ≥ 4 mice per condition, and n> 10 sections per animal (H) qPCR levels of LOX-L2 and HIF-1A in PSCs, normalized to RPLP0 and relative to 1 kPa (I) qPCR levels of LOX-L2 and HIF-1A in PSCs, normalized to RPLP0 and relative to control (J) Quantification of average forces applied by PSCs on pillars. BBI = blebbistatin. In the box and whisker plot, the central box represents values from the lower and upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum. ***P<0.001 Three experimental repeats (K) qPCR levels of LOX-L2 in PSCs, normalized to RPLP0 (60S acidic ribosomal protein P0) and relative to control
Figure 7FAK-expressing SCC cells were grown on glass coverslips, fixed and stained with anti-FAK anti-Paxillin and DAPI Representative immunofluorescence images are shown. Scale bars, 20 μm. B FAK-expressing SCC cells were grown on glass coverslips, fixed and stained with anti-pSrc Y416 , anti-Paxillin and DAPI Representative immunofluorescence images are shown. Scale bars, 20 μm. The graph (B) shows the quantification of internalized active Src from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars, s.d. * = p < 0.01. C Subcellular fractionation of SCC cells expressing FAK-WT or interface mutant FAK. Whole cell lysates (WCL) and subcellular fractions were subjected to western blot analysis with anti-FAK, anti-Tubulin (marker for cytoplasmic fraction; C), anti-RCAS (marker for perinuclear fraction; PN) and anti-H4 (marker for nuclear fraction; N). The graph shows densitometric analysis of relative FAK amount of the FAK mutants in the different fractions from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars: s.d. * = p < 0.01, # = p < 0.05. For full blots see source data.
Tubular LC3 expands along microtubules.Figure 3.(A) The indicated cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr followed by immunofluorescence microscopy with anti-Cytochrome c antibody. Confocal images were acquired as z-stacks comprising 6 sequential sections with 0.8 μm z-intervals. Scale bars, 10 μm. (B) TBC1D15−/− cells prepared as in (A) were subjected to immunoelectron microscopy with anti-GFP antibody. White arrowheads indicate YFP-LC3-labeled tubules. High magnification image is shown in the lower panel. Scale bars, 500 nm. (C) The indicated cells prepared as in (A) were subjected to immunostaining with anti-Tubulin antibody. YFP-LC3 and Tubulin staining are merged in the right panels. Magnified images are shown for TBC1D15−/− cells. Scale bars, 10 μm. (D) The indicated cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin in the presence or absence of nocodazole for 3 hr. YFP-LC3 and mCherry-Parkin images are merged in the right panels. Scale bars, 10 μm. (E) YFP-LC3 morphologies of cells in (D) were quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.
In vivo luminescence imaging of mice after intravenous injection of brain-enriched AAV2 vectors carrying a luciferase reporter gene under control of the CAG promoter.Figure 2A Luciferase reporter gene vectors displaying the brain-targeted peptide NRGTEWD ("BR1"), unmodified wild type AAV2 capsid, or the previously reported brain-targeting peptide DSPAHPS (PPS). Vectors were intravenously injected into mice (5x1010 genomic particles/mouse). Panels show representative examples of n = 5 animals, age 8-12 weeks per group. Animals were imaged in dorsal (left panel), ventral (second from left panel), and lateral (second from right and right panel) position, 14 days after vector injection.B Close-up imaging of AAV-BR1-treated mice. Mice were imaged in dorsal and lateral position in a different color scheme allowing detailed visualization of the transduced brain in living animals, even as late as at day 264 after vector injection.C Virtual sections : sagittal, coronal, and transaxial (left panels) and three-dimensional reconstruction (right panel) of the luminescence images of a mouse injected with BR1 vector (as in A). Images were obtained by measuring different wavelengths of the emitted light (Living Image software), confirming the brain as exclusive source of luminescence.