EMBO/sd-character-level-ner huggingface dataset:
Prazosin induces GIC apoptosis in a PKC-dependent manner.Figure 5A. PKCδ expression analysis in two patient-derived GICs and NSCs by immunoblotting. GICs express higher levels of PKCδ than NSCs. kDa: kilodaltons. B. PKCδ by immunostaining (green). Nuclei were stained with DAPI (blue). Enhanced PKCδ punctate nuclear expression in prazosin-treated (PRZ, 5µM) GICs, as compared to vehicle-treated GICs. PRZ: prazosin. Scale bar: 10 µm. C. Total PKCδ (PKCδ 78kDa) and cleaved PKCδ (PKCδ 41kDa) expression analysis in GICs by immunoblotting. Prazosin (PRZ) induces PKCδ cleavage into a 41kDa active fragment. 2 µM rottlerin (Rott), a PKCδ inhibitor, inhibits prazosin-induced PKCδ cleavage. V: vehicle. kDa: kilodaltons. D. Pro-caspase-3 (Pro-CASP3) and activated caspase-3 (CASP3) expression analysis in GICs by immunoblotting. 2 µM rottlerin (Rott) inhibits prazosin (PRZ)-induced caspase-3 activation. V: vehicle. kDa: kilodaltons. E. Inhibition of PKCδ by rottlerin prevents, in a concentration-dependent manner, prazosin-induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of rottlerin. *P=0.0286, n=4, two-sided Mann-Whitney U-test. F. Inhibition of PKCδ by the specific anti-PKCδ RACK peptide δV1.1 (10 μM) counteracts prazosin-induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of δV1.1. *P < 0.005 for Prazosin 1 and 5 µM and P<0.001 for Prazosin 10 µM by two-tailed unpaired Student's t-test, n=4. G. Decreased PKCδ expression using shRNA counteracts prazosin-induced GIC death. Viability analysis of GICs transduced with scrambled or PKCδ shRNA and treated with prazosin for 72 h. *P < 0.005 by two-tailed unpaired Student's t-test, n=4. H. Prazosin inhibits AKT phosphorylation in GICs. LY294002 (LY, 30 µM), an inhibitor of the PI3K/AKT pathway, was used as positive control. Terazosin, which does not affect GIC viability, does not alter AKT phosphorylation (see the corresponding cell viability counting in Fig EV4C). Phosphorylated AKT (P-AKT) and total AKT (AKT) expression analysis in GICs by immunoblotting. V: vehicle. kDa: kilodaltons. I. Prazosin (10 µM) does not inhibit AKT phosphorylation in NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons. J. β-catenin expression, a downstream target of AKT, is decreased in prazosin-treated GICs, as opposed to NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons. K. L. Inhibition of PKCδ using rottlerin (Rott, 2 µM) or δV1.1 (10 µM) counteracts prazosin inhibition of AKT phosphorylation. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.
Figure 3A Cholesteryl ester synthesis, as measured by 3H-oleate incorporation. Note increased CE in ApoE4-treated cells, whereas there was essentially no difference in triglyceride (TAG) production (mean±SE; n=3. with 3 replicates/experiment). †, p=0.06 vs E3. B Comparison of 3H-oleate incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 5 replicates/experiment). C ApoE ACM-treated fibroblasts were stained with LipidTox green neutral lipid stain, and lipid droplets were visualized (example at left) and counted (right). Note significantly more lipid droplets in ApoE4-treated cells (mean ± SD, n=3).
Mito-C causes DRP1 dependent mitochondrial fission and ER-mitochondria contact sites increaseA, HeLa cells treated or not, with 2µM Mito-C for 15 min were immunostained with anti-TOM20 (red) and anti-DRP1 (green); arrowheads in the far-right panel indicate recruitment of DRP1 onto the mitochondrial surface (TOM20).Figure 3A, HeLa cells treated or not, with 2µM Mito-C for 15 min were immunostained with anti-TOM20 (red) and anti-DRP1 (green); arrowheads in the far-right panel indicate recruitment of DRP1 onto the mitochondrial surface (TOM20). B, Quantification of DRP1 signal on TOM20 positive structures. Errors bars show the standard error of the mean (SEM) (n=45, technical replicates). C C, Western blot analysis of DRP1, GAPDH (cytosolic marker) and TOM20 (mitochondrial marker) protein in cytosolic and mitochondrial fractions treated or not, with 2µM Mito-C as indicated. D, Quantification of western blots showed in C and expressed as a distribution of DRP1 in the cytosolic and mitochondrial fractions. Errors bars show the standard deviation (SD) of 5 independent experiments. E E, HeLa cells were transfected with DRP1K38A mutant and mCherry and treated or not, with Mito-C (T for transfected, NT for not transfected). F, Quantification of the mitochondrial phenotypes observed in E. Errors bars show the standard error of the mean (SEM). (30-35 images each with an average of 15-20 cells from triplicate independent experiments were analyzed). G G, Electron micrographs (EM) from HeLa cells treated with 2µM Mito-C or DMSO for 120 minutes. Empty arrowheads indicate ER-mitochondria contact sites. H, Quantification of ER-mitochondria membrane contact sites events (reported in 50µm² section; 50 images from an average of 30 cells from triplicate independent experiments were analyzed). Errors bars show the standard error of the mean (SEM). I, Quantification of ER-mitochondria membrane contact sites density (in µm; 30 images from an average of 30 cells from triplicate independent experiments were analyzed). Errors bars show the standard error of the mean (SEM). Da
a) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2 (no.figf7(a) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2 (no. 1 or 2) or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition. (b) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2, both FBXL2 and p85β, or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition. (c) U2OS cells stably transfected with a doxycycline-inducible p85β construct were serum starved for 48 h. During the last 16 h before collection, p85β expression was stimulated with doxycycline. Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition. (d) Cells constitutively expressing GFP-LC3 were transfected with HA-tagged p85β, fixed and incubated with an anti-HA p85β antibody (red). Arrows in the top panels point to the enhanced autophagic response (LC3-II vesicles) in cells expressing high levels of p85β, as shown in the bottom panels. Uncropped images of blots are shown in Supplementary Fig. S8. Scale bars, 10 μm.
a) Ubiquitylation of Dvl2 enhanced by ectopically expressed pVHL.figf6(a) Ubiquitylation of Dvl2 enhanced by ectopically expressed pVHL. (b) In vitro ubiquitylation of Dvl2 by the pVHL E3 ligase complex including pVHL, Elongin B, Elongin C, Rbx1 and Cul2. In vitro-translated Flag-Dvl2 was incubated with or without purified E1, E2 and E3 ligase complex, Elongin C and pVHL. The reaction was subjected to anti-Flag immunoprecipitation. Dvl2 ubiquitylation was examined by using anti-ubiquitin immunoblotting. (c) Ubiquitylation of Dvl2 is decreased by pVHL knockdown. (d) Starvation elevates Dvl2-Cul2 interaction. After co-transfection with Myc-Cul2 and Flag-Dvl2 for 36 h, HEK-293T cells were cultured in nutrient-rich medium or starved with BFA1 (0.1 μM) for 4 h. Cells were harvested for anti-Myc immunoprecipitation followed by anti-Flag immunoblotting. Total protein expression was confirmed by immunoblotting with whole-cell lysates. (e) pVHL knockdown or UbKOGA blocks Dvl2 binding with p62 under starvation conditions. (f) Dvl2 ubiquitylation is required for its direct interaction with p62. Dvl2 was ubiquitylated by the pVHL E3 ligase complex in vitro, followed by in vitro binding assay with recombinant p62. (g) pVHL knockdown decreases Dvl2 degradation under starvation conditions. (h) Knockdown of p62 or LC3 attenuates the pVHL-promoted Dvl2 degradation under starvation conditions. Uncropped images of blots are shown in Supplementary Information, Fig. S8.
Figure 1(A) Analysis of the single-cell transcriptomics dataset for the mouse visual cortex (Tasic et al, 2016) revealed that the expression of the metabolism-associated gene Cox6a2 is highly restricted to PV+ interneurons. Cox6a2 is expressed in all subtypes of PV+ interneurons (marked in blue color) but rarely detected in other neuronal subtypes from the mouse visual cortex. Cox6a1, which encodes the other protein isoform of COX6A, is expressed in all subtypes. (B RT-PCR showing expression of Cox6a2 in the brain and heart. C) Western-blot showing expression of Cox6a2 in the brain and heart. (D) Western-blot analysis showed that COX6A2 is first detected in the brain at postnatal day 0 (P0). Then, COX6A2 protein levels gradually increase until P60. COX6A2 immunoreactivity is detected in PV+ interneurons in different brain areas of adult mice, including the cortex (general overview in E, and single PV+ interneuron magnification in F) White arrowheads indicate PV/Cox6a2 double positive cells. COX6A2 immunoreactivity is detected in PV+ interneurons in hippocampus (G) and striatum (H). White arrowheads indicate PV/Cox6a2 double positive cells. (I,J) Quantifications showing almost 100% of co-localization of COX6A2+ cells and PV+ cells in the somatosensory (SS) and motor (MT) cortices, and the hippocampus (HI) of adult mice (n animals=4, 2 months old, n sections=24 SS, 16 MT, 8 HI). (K) COX6A2 immunoreactivity in the cortex at P9 (n animals=2, n sections=12 SS, 8 MT, 4 HI). White arrowheads indicate Cox6a2 positive cells. (L-N) As soon as parvalbumin immunoreactivity becomes visible at P13, almost 100% of PV+ cells are co-labelled with COX6A2 immunoreactivity in the SS and MT cortices, and the HI of P13 mice (n=2).
Figure 2Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls. Two days later, cells were subjected to immunoblotting (A Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls. Two days later, cells were subjected t flow cytometry to quantify the percentage of GFP positive cells (B The GFP quantification in (B) displays averages (bars) of 3 independent experiments (dots) Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls Two days later PI-stained cellular DNA (C) Co-depletion of Wapl and separase has a synergistic effect on HDR. U2OS DR-GFP cells were transfected with the indicated siRNAs, treated as in (A+B) and analysed by GFP-flow cytometry (D Shown in (D) are averages (bars) of 2-3 independent experiments (dots) Co-depletio of Wapl and separase has a synergistic effect on HDR. U2OS DR-GFP cells were transfected with the indicated siRNAs, treated as in (A+B) and analysed b immunoblotting (E)
Figure 7Cartoon summarizing the current findings. RapZ, presumably in its tetrameric state, binds GlcN6P in its CTD. Upon GlcN6P scarcity, RapZ accumulates in its "free" form and activates phosphorylation of QseE/QseF by direct interaction. Activity of the TCS depends on lipoprotein QseG (Göpel & Görke 2018), suggesting that RapZ can only activate those kinases, which are contacted by QseG in the periplasm. QseF~P triggers glmY expression from its σ54 promoter augmenting levels of GlmY*, which subsequently sequesters RapZ into stable complexes. Consequently, RapZ is not available to trigger decay of sRNA GlmZ, which therefore activates synthesis of GlmS, replenishing GlcN6P. Sequestration also precludes RapZ from activating QseE/QseF, providing a negative feedback loop that adjusts GlmY amounts to the required level. GlcN6P releases GlmY* from RapZ, which is then free to promote GlmZ decay repressing glmS.
Identification of direct PBX1 target genes.Figure 5(A) Chromatin immunoprecipitation sequencing (ChIP-seq) identified a PBX1 binding core sequence motif composed of a 10-nucleotide motif (A) and a 5-nucleotide motif (B). (B) Schematic of the track of PBX1 ChIP-seq compared to IgGChIP-seq as a control on different loci with peaks identified by HOMER. Each locus represents a 20kb chromosome region. (C) List of the 29 most regulated genes (t-test q-value <0.05) identified by Gene Set Enrichment Analysis (GSEA) and ranked by p-value. RPKM stands for Reads Per Kilobase of transcript per Million mapped reads. GSEA was performed on genes identified by RNA-Seq as differentially expressed in the VM at E12.5 compared to adjacent brain regions and identified by PBX1Chip-Seq in proximity to transcription starting sites. (D) Graphic representation of these top 29 genes (red dots) in the GSEA curve showing a high correlation between the cistrome and transcriptome (p-value <0.001, q-value<0.005). (E) ChIP followed by PCR confirmed that PBX1 binds to genomic regions in close proximity to the TSS of Pitx3 (Pitx3-Gbf1), Onecut2and Nfe2l1 genes. No co-immunoprecipitation was detected for the unrelated gene, Lrp1b (Low Density Lipoprotein Receptor-Related Protein 1B). (F, G) The regulation of Pitx3 and Onecut2 by PBX1 was confirmed in Pbx1-/-;Pbx3+/- mutant embryos at E12.5, compared to wild-type (Pbx1+/+) littermates. (F) Representative immunofluorescence microscopy/confocal images showing that decreased levels of PITX3 and number of PITX3+cells in E12.5 Pbx1-/-;Pbx3+/- embryos. This reduction is more evident in the basal plate (BP) and in the midline (M), as shown at higher magnification (right). (G) Double ONECUT2+TH+cells were detected only in the lateral part of the mFP in E12.5 Pbx1-/-;Pbx3+/- embryos. Selected areas are magnified to the right. A scheme (right) shows the distribution of cells in Pbx1+/+ and Pbx1-/-;Pbx3+/- embryos. All scale bars, 40µm. Error bars represent mean ± standard deviation. Biological replicates (dots) per condition and p-value (T-test) are indicated in each graph.
Figure 3(A and B) Scatter plots showing the connection between KDM7A or UTX binding and RNA expression in TNF-α-stimulated ECs. The X and Y axes indicate the log2 ratio of fragments per kilobase of exon per million mapped sequence reads (FPKM) values and the ChIP-seq signals (TNF-α 60min/0min) for KDM7A (A) and UTX (B), respectively. Genes with more than a 1.5-fold increase in FPKM values (light red dots) satisfying a 0.5-fold increase in ChIP-seq signals are indicated as dark red dots. Genes with a 1.5-fold decrease in FPKM values (light blue dots) satisfying a 0.5-fold decrease in ChIP-seq signals are indicated as dark blue dots. These genes are defined as the target genes for KDM7A (A) and UTX (B), respectively. The names of representative genes are shown in the graphs. (C and D) Metagene representations of KDM7A (C) and UTX (D) ChIP-seq signals in units of read count per million mapped reads at a meta composite of the genomic regions around the TNF-α-up-regulated genes. (E) De novo motif analyses for KDM7A (top) and UTX (bottom) binding recognition sequences in TNF-α-stimulated ECs. The P-value indicates significant enrichment of the transcription factor binding motif in KDM7A/UTX binding sites, in comparison to a size-matched random background. (F) Heat map of H3K27ac (green), p65 (yellow), KDM7A (red), and UTX (purple) levels in resting and TNF-α-stimulated ECs. Each row shows ± 10 kb centered on the KDM7A (top) or UTX (bottom) peak. Rows are ordered by max KDM7A or UTX signal in each region. The ChIP-seq signal is depicted by color scaled intensities. (G and H) Gene tracks of ChIP-seq signals for H3K27Ac, p65, KDM7A, and UTX, and RNA-seq signals around the VCAM1 (G) and SELE (H) loci in TNF-α (-) and TNF-α (+) ECs. ChIP-Seq and RNA-seq signals are visualized by Integrated Genome Viewer (http://software.broadinstitute.org/software/igv/). (I and J) ChIP-qPCR of p65 (I) and KDM7A (J) at the VCAM1 TSS, normalized to input. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). † P < 0.05 compared to TNF-α (+) siKDM7A (-). Statistical differences were analyzed by the Tukey-Kramer test. (K and L) ChIP-qPCR of p65 (K) and UTX (L) at the SELE TSS, normalized to input. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Tukey-Kramer test.
Figure 3Time-lapse Airyscan imaging of ΔΨm in living HeLa cell, co-stained with MTG (ΔΨm-insensitive after loading) and TMRE (ΔΨm sensitive). Arrowhead points to a flickering event where a mitochondrion depolarizes (~9 sec) and repolarizes (~16 sec), showing that heterogeneous signal from TMRE (but not MTG) disappears and reappears. Scale bar = 500 nm. N = 4 independent experiments. Quantification of A. Plot shows average TMRE fluorescence intensity (FI) of cristae (dark red line) vs. matrix (light red line) during the time series. The drop in TMRE FI during the depolarization phase of the flickering is the ΔΨm-sensitive component of the TMRE signal. The remaining TMRE FI during depolarization can be considered as the ΔΨm-insensitive portion of TMRE signal. Note that the remaining TMRE FI after depolarization at the cristae and matrix are approximately identical, indicating that differences in TMRE FI between the cristae and matrix prior to depolarization are derived from differences in ΔΨm. N = 4 independent experiments.
Figure 1A. qRT-PCR analysis of RNA samples from normal human skin (NS) and AK for activin βA (INHBA), normalized to hypoxanthine phosphoribosyltransferase (HPRT). Expression levels in one of the normal skin samples were set to 1. Scatter plots are shown. NS: N=4; AK: N=21. ns = non-significant (Mann-Whitney test). B. qRT-PCR analysis of RNA samples from normal back skin of wild-type mice (N=4, n=4), non-tumorigenic back skin from HPV8/wt mice (N=7, n=9) and papillomas from HPV8/wt transgenic mice (N=8, n=11) for Inhba, normalized to glyceraldehyde 3-phosphate dehydrogenase (Gapdh). Expression levels in one of the wild-type back skin samples were set to 1. Statistical significance was determined using 1-way ANOVA and Bonferroni`s Multiple Comparisons Test of log-transformed data. N, number of mice; n, number of biopsies. C-E. Sections from a tumor collected from a 64 week-old HPV8/wt mouse (C,D) or from non-tumorigenic skin of 5 week-old HPV8/wt mice (E) stained with an anti-activin A antibody and counterstained with hematoxylin. E: Epidermis, D: Dermis, HF: Hair follicle. (D) Shows a higher magnification of the boxed area in (C). Scale bars: 200μm (C) and 100μm (D,E). F. Kinetics of tumor incidence in HPV8/wt and HPV8/Act mice; N=20 mice per genotype. Statistical significance was determined using Log-rank (Mantel-Cox) test. G. Age of the mice when the first tumors appeared on certain body sites. Each dot represents one tumor. H. Representative macroscopic pictures of ear tumors (left panel) and H&amp;amp;E-stained sections of ear acanthopapillomas (upper and middle panel) or an ear acanthopapilloma with trichoepitheliomatous differentiation (lower panel). Boxed areas are shown at higher magnification in subsequent pictures to the right in each horizontal picture panel. E: epidermis; D: dermis; C: cartilage. Scale bars: 200μm (left panel), 100μm (middle panel) or 50μm (right panel).
AMPK interacts with ULK1 directly.Figure 1(A) 293T cells were transfected with Flag-ULK1 or control empty vector and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblot analysis with the indicated antibodies. (B) 293T cells were co-transfected with Flag-AMPKα2 and HA-ULK1. Cell lysates were subjected to immunoprecipitation using anti-Flag antibody followed by SDS-PAGE/immunoblot analysis with anti-HA and anti-Flag antibodies. (C) 293T cells were transiently transfected with Flag-AMPKα2 or Flag-AMPKβ1. After 24 h, immunoprecipitation was performed using anti-Flag or control anti-HA antibodies and analyzed by immunoblotting with anti-ULK1 and anti-Flag polyclonal antibodies. (D) 293T cell lysates were subjected to immunoprecipitation with anti-ULK1 polyclonal antibody or control preimmune rabbit serum (NRS), followed by immunoblot analysis with anti-ULK1 and anti-AMPKα antibodies. (E) 293T cells were infected with ULK1 shRNA or control shRNA lentiviruses and subjected to immunoprecipitation with control rabbit IgG or anti-AMPKα antibody followed by immunoblotting with anti-ULK1 and anti-AMPKα antibodies. (F) Purified His6-AMPKα1/β1/γ1 fusion proteins (250 ng) were mixed with purified His6-tagged ULK1 (1 µg) or Bcl-XL (200 ng) proteins in 1% Triton X-100 lysis buffer containing protease inhibitors and subjected to immunoprecipitation with anti-ULK1 antibody. The resulting protein complexes and 10% of the input proteins were analyzed by immunoblotting with anti-His-Tag polyclonal antibody.
Figure 6HBO1, as part of two distinct KAT complexes, occupies the chromatin environment surrounding a large fraction of replication origins found close to TSSs. The BRPF3 scaffold directs HBO1 specificity towards H3K14ac, which in turn facilitates the activation of replication origins. Previous work has shown that the HBO1-JADE complex promotes H4K5/8/12 acetylation and facilitates MCM2-7 loading (Miotto Struhl, 2008; Miotto Struhl, 2010). Thus by partnering with different scaffolds, HBO1-mediated acetylation of chromatin facilitates two consecutive steps, licensing and activation, in replication initiation.
Simulation of the steady-state response curves of glucose uptake rate, energy charge, and ATPase flux under anaerobic growth for increasing ATPase activities.Figure 6A) Simulations with the standard kinetic model (version 1). The measured glucose uptake rates of the different strains are indicated B) Simulations with the kinetic model (version 1) as in (A), but without ADP activation term in the PFK kinetics.
Figure 6Dephosphorylation of Rad53 after exposure to HU. WT, rpb1‐1 and rpb1‐S751F cells were synchronized in G1 with α‐factor. After G1 release, cells were exposed to 100 mM HU for 1 h, washed to remove HU and cultured in HU‐free medium for the indicated periods. Western blot against Rad53, signal quantification (ratio Rad53‐P/total Rad53 protein) and FACS profiles are shown. DNA repair of MMS damage. Chromosome VII species revealed by hybridization with ADE5,7 probes in pulsed‐field gel electrophoresis (PFGE). Asynchronously growing WT and rpb1 cells were exposed to 0.1% MMS for 30 min, washed to remove MMS and cultured in MMS‐free medium for the indicated periods to allow repair. Repair was calculated as the percentage of FLC appearing after MMS treatment. The FLC signal was quantified with respect to the total signal of each lane. Data show the mean and SD of 3 independent experiments. NLC, nonlinear chromosome. FLC, full‐length linear chromosome. CF, chromosome fragment resulting from DNA breaks.