EMBO/sd-character-level-ner huggingface dataset:
Figure 6Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Scheme of the experiments. Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Survival was monitored daily for 2 weeks. Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Two days post-infection, virus titers in lung homogenates were determined by plaque assay. A p value of <0.05 was considered significant (** p < 0.01). Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Two days post-infection, the expression level of IFNβ in the lungs was analyzed by semi-quantitative RT-PCR (left panel) and qPCR (right panel). A p value of <0.05 was considered significant (** p < 0.01). E Proposed model for innate immune evasion by the 1918 pandemic virus.
Figure 5(A) Substrate binding was assayed by anisotropy using a fluorescently labeled substrate peptide. All SPOP proteins bound substrate with an affinity similar to that of SPOP28-359 (4-8 µM, see Table 2). Experimental data are shown as circles; the solid lines are fits to eq. 2 (Roehrl et al, 2004). (B) In vitro crosslinking assays were performed for SPOP28-359 and each mutant at 30 μM protein with the amide-specific BS3 crosslinker. Crosslinking for SPOP ΔBACK and MATH domain are shown to demonstrate that crosslinking conditions do not lead to non-specific crosslinking of protein species. (C) Crosslinking reactions were performed on whole cell lysates from cells expressing wild type SPOP, SPOP mutBACK, SPOP mutBTB, or SPOP mutBTB-BACK. SPOP1, SPOP2 and SPOPn identify SPOP monomers, dimers and larger species, respectively. For loading levels see Appendix Fig S4.
a) Beclin 1 or Atg14L siRNA reduced Atg14L levels and increased p62/SQSTM1 and LC3 II levels under normal and nutrient-starvation conditions in NIH 3T3 cells.figf2(a) Beclin 1 or Atg14L siRNA reduced Atg14L levels and increased p62/SQSTM1 and LC3 II levels under normal and nutrient-starvation conditions in NIH 3T3 cells. (b) Compared with control siRNA, Atg14L siRNA decreased long-lived protein degradation in NIH 3T3 cells under normal (*P = 0.007) and starvation (*P = 5 × 10−6) conditions (one-tailed Student's t-test with equal variances, n = 4). This difference was diminished when the starved cells were treated with 3-methyladenine (3MA, 10 mM), a PI(3) K inhibitor. (c) Vps34 kinase assay. HEK 293T cells were co-transfected with Myc-Vps34-Vps15 and Flag-Atg14L or Flag vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by anti-Myc antibody for the in vitro kinase assay. The resulting radioactive PI(3) P was separated by thin-layer chromatography (TLC), quantified and normalized against the amount of immunoprecipitated Myc-tagged Vps34 as measured by western blot (upper panel). The quantified results (lower panel) show that overexpressing Atg14L significantly upregulated Vps34 kinase activity by 2.5-fold, but only when Beclin 1 was also overexpressed (*P = 0.04, one-tailed Student's t-test with unequal variances, n = 5). (d) Colocalization of co-expressed Atg14L-EGFP (green) and Beclin 1-AsRed (red) in punctate structures in transiently transfected HeLa cells. Scale bar, 10 μm. (e) Electron microscopy images show large structures (asterisks) that are often enwrapped with double membranes in the HEK 293T cells co-transfected with Atg14L-EGFP and Beclin 1-AsRed: concentric membrane 'rings' (panel 1); two large structures (3-5 μm in diameter, panel 2) containing material with high electron density (inset, enwrapping double membranes); numerous autophagosomes (arrows, panel 3) in the cytoplasm; immuno-electron microscopy image of a Atg14L-Beclin 1 structure (labelled with anti-GFP antibody and developed by DAB, panel 4) enwrapped with concentric membrane 'rings'. M, mitochondria; N, nucleus. Scale bar, 500 nm. (f, g) EGFP-Atg12 (f) or EGFP-Atg5 (g) (green) was colocalized with the large structures (arrows) that were labelled by Atg14L-AsRed (red) and Beclin 1-Myc (blue) in transfected HeLa cells. Some of these structures seemed to be 'ring'-shaped (yellow arrows and inset). Scale bar, 10 μm. See Supplementary Information, Fig. S6 for full scans of blots in a and c.
a) RPE cells transiently transfected with siRNA against myosin VI and Atg5 were treated with 1 μM MG132 for 16 h.figf2(a) RPE cells transiently transfected with siRNA against myosin VI and Atg5 were treated with 1 μM MG132 for 16 h. Cells were processed for immunofluorescence microscopy following 16 h MG132 treatment (zero time point) and 8 h post-washout of inhibitor. Immunolabelling for p62 was performed to visualize aggregates (red) and Hoechst was used to identify nuclei (blue). (b) Top, western blot analysis confirming siRNA depletion. Bottom, quantification of immunofluorescent p62-positive aggregates was evaluated using an automated Cellomics VTi microscope. The results were calculated as the average p62 fluorescence intensity at 8 h post-MG132 washout normalized to the zero time point and are represented as the percentage of the control (±s.d.; n = 3). *P0.05, ***P0.001. See Methods and Supplementary Table S1 for details of the SMARTpool (SP) and single (07) oligonucleotides. (c) Parental HeLa cells or HeLa cells stable expressing siRNA-resistant GFP-myosin VI were transiently transfected with a single siRNA oligonucleotide targeting myosin VI and were subsequently treated with 1 μM MG132 for 16 h. Cells were processed for immunofluorescence microscopy at 16 h post-MG132 treatment (t = 0) or allowed to recover following washout of MG132 for 8 h (t = 8). Cells were processed for quantification with the automated Cellomics VTi microscope to evaluate the p62 fluorescence intensity. The results represent the fold increase in p62 fluorescence intensity of myosin VI siRNA compared with mock control cells following recovery from MG132 washout (t = 8; ±s.d.; n = 3). (d) HeLa cells with stable expression of HttQ72-GFP were transiently transfected with siRNA against myosin VI followed by saponin extraction and processing for immunofluorescence microscopy. Immunolabelling was performed for GFP (green) and p62 (red). Nuclei are labelled with Hoechst (blue). (e) Quantification of HttQ72-GFP aggregates was performed on myosin-VI-siRNA-transfected HeLa cells. The results were calculated as the percentage of GFP-expressing cells with greater than 15 GFP-positive spots per cell. The results represent the mean (±s.d.) from n = 3independent experiments, ***P0.001. The insets show higher magnifications of the areas outlined in the main images. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.
Figure 6Representative examples of triple fluorescent whole mount WM-ISH labelling of gfap (green), elavl3 (magenta) and her6 (grey) domains of expression (top panels) in hindbrain rhombomere6 (r6) in wild-type embryo observed at 34hpf; merged images indicate how the her6 expression domain overlaps with the progenitor zone (NP=gfap(+)/elavl3(-)), transition zone (T=gfap(+)/elavl3(+)) and neurogenic zone (N=gfap(-)/elavl3(+)); (bottom-right panel) schematic representation showing the her6 domain spanning the NP, T and N zones with quantification of distances from dorsal; transversal view; annotations denote dorsal (D) and ventral (V); scale bar 30μm. Single molecule fluorescent in situ hybridization (smFISH) showing her6 (green), elavl3 (magenta) and dapi nuclear staining (blue) obtained from hindbrain (r6) sections of wild type embryo at 34hpf; head arrows indicates examples of co-existence of transcriptional active sites for her6 and elavl3; scale bar 3μm. Triple fluorescent WM-ISH for gfap (green) and elavl3 (magenta) and her6 (grey) in the hindbrain (r6) of CTRL (top) and MBSm (bottom) embryos at 52hpf; dorsal (D); ventral (V); scale bar 30μm. Magnification of inset from merged gfap/elavl3 in (c) showing CTRL (top) and MBSm (bottom) embryos with corresponding NP/T/ N and NP/T zones respectively; dorsal (D); ventral (V); scale bar 30μm. Normalized intensity mean of elavl3 and gfap along the DV axis spanning the NP/T/N zones in CTRL and NP/T zones in MBSm respectively; region of interest (ROI) delineates high elavl3 versus gfap in CTRL (T and N zones) while in MBSm only the T zone is observed, overlap of elavl3 and gfap intensity mean peaks. Normalized mean of elavl3 and gfap intensities in ROI observed in: (f) uninjected (UI) (5 embryos, 62 slices), (g) control (CTRL) (5 embryos, 35 slices) and (h) MBSm (8 embryos, 62 slices) conditions; bars represent mean and SEM; multiple t-test with Benjamini, Krieger and Yekuteli discovery, significance: p<0.05*, p<0.001***, p<0.0001****. Transverse sections of double fluorescent WM-ISH for gfap (green) and neuroD4 (blue) in the hindbrain (r6) of CTRL (top) and MBSm (bottom) embryos at 52hpf; dorsal (D); ventral (V); scale bar 30μm. Chromogenic WM-ISH showing neuroD4 expression intensity in: uninjected (UI) 51/56 embryos (91%), CTRL 50/54 embryos (93%) and mutant (MBSm); 36/65 embryos (55 %); longitudinal view, scale bar 100μm.
Figure 4This model is obtained by superimposition of FGF21core structure with the crystal structures of β-klotho/FGF21-CT (PDB ID: 5VAQ), FGF23/FGFR1c/α-klotho (PDB ID: 5W21) and FGF2/FGFR1c/HS dimer (PDB ID: 1FQ9), and is shown as surface representation. FGF21core is colored orange. β1-β2 loop of FGF21 (Asp24 to Ala31) directly interacting with FGFR1c is colored magenta. FGF21CT binding to β-klotho is colored blue. FGFR1c is colored green. D1, D2 domain and the receptor binding arm of β-klotho are colored cyan, lightblue and pink, respectively.
a) NRK cells stably expressing CFP–LC3; LAMP1–YFP were transfected with nonspecific (NS)- or AP4-RNAi and starved for 12 h.figf6(a) NRK cells stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or AP4-RNAi and starved for 12 h. Scale bars, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d. (c) Representative TEM micrographs of nonspecific- or AP4-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bars, 5 μm. (d) NRK cells were starved for 0, 4, 8 or 12 h, and isolated lysosomes/autolysosome were analysed by western blotting using antibodies against AP4 or LAMP2. (e) Purified reformation tubules were stained with antibodies against AP4 and LAMP1. Scale bar, 2 μm. The bottom row shows enlarged regions of interest from the top panel. (f) NRK cells were transfected with GFP and LAMP1-GFP. At 20 h after transfection, cells were starved for 0 or 8 h, and then collected and analysed by immunoprecipitation (IP) and western blotting (WB) of proteins as indicated, using stably expressed LAMP1-GFP as bait. TCL, total cell lysate. The insets in a, c and g represent enlarged regions of interest. (g) NRK cell lines stably expressing LAMP1-Cherry; clathrin-GFP were transfected with nonspecific- or AP4-RNAi and starved for 12 h. Scale bars, 5 μm. (h) Cells from g were analysed for the Pearson's co-localization coefficent (R(r)) by Olympus Fluoview. n = 30 cells from three independent experiments. Error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.
Multi-faceted system-level analyses identifies seven prostate cancer hub genes.Figure 1Module-trait relationships were established by WGCNA using eight independent microarray analyses comprising 375 human prostate samples. Gene modules (y axis) are denoted by an arbitrary color name. Bins show the Pearson correlation value between gene expression levels of each module within the noted phenotype/disease stage (x axis) and p values. A value of 1 (red) quantifies the strongest positive correlation (genes are upregulated), -1 (blue) the strongest negative correlation (genes are downregulated), and 0 (white) no correlation. Microarray analysis was performed following doxycycline-regulated specific AR-V7 depletion (using a tet-plKO backbone) in 22Rv1 PC cells compared to doxycycline-treated shGFP controls. The genes that were significantly regulated by shAR-V7 (in either direction, p value < 0.05) were distributed among the gene modules defined by WGCNA in panel A. Upregulated genes (red) are those in which expression decreased following AR-V7 depletion and conversely downregulated genes (blue) are those that increased following AR-V7 depletion. AR-V7 human functional interactome was generated using SGA screening in the yeast S. pombe, combined with STRING data to map protein-protein interactions, followed by the identification of the human orthologs. The colors denote the different types of genetic interactions: red are genes that when deleted in yeast and crossed with AR-V7-expressing yeast were synthetic lethal while green denotes genes that caused a growth suppression. White designates yeast essential genes (ie: genes that are critical for yeast survival, and thus could not be present in the yeast deletion library), but were incorporated into the network based on the criteria that they are known (based on literature) to physically interact with at least two of the red or green genes. Pink are a combination of essential and nonessential genes identifying the same human protein. Table summarizing the seven PC hub genes identified by the system-level analyses. Network interactions of the seven genes with the 50 most frequently altered neighbor genes, were mapped using cbioportal.org. The type of gene to gene interactions are: controls state change of (blue), controls expression of (green), in complex with (brown).
siRNA knockdown of pulmonary vascular ISCU1/2 in vivo promotes PH.Figure 7A Schema of the strategy for pharmacologic inhibition of siRNA knockdown of ISCU1/2 in the pulmonary vasculature of mice in vivo by Staramine-mPEG-mediated intravenous delivery. B, C Pharmacologic inhibition of ISCU (N = 8/group) did not influence miR-210 expression in the lungs either after Norm + SU5416, NS P = 0.8634 (B) or Hyp + SU5416, NS P = 0.9786 (C). D As compared with control siRNA (siCont) where ISCU1/2 expression was decreased after exposure to Hyp + SU5416 (white bars), lung delivery of siRNA specific for ISCU1/2 (siISCU) via Staramine-mPEG nanocomplexes (Polach et al, 2012) down-regulated ISCU1/2 in < 100-μm pulmonary vessels in Norm + SU5416 and Hyp + SU5416 (N = 5/group, ***P = 0.0006, **P = 0.0019 for Hyp + SU5416, **P = 0.0029 for siCont). E By electron paramagnetic resonance (EPR) spectroscopy (representative siCont versus siISCU lung), Fe-S cluster signal was decreased (quantitative graph, right) in siISCU mouselung tissue (N = 4 mice) compared with siCont (N = 5 mice), *P = 0.0143. F Endothelin-1 was increased in lung tissue after siISCU delivery under normoxic conditions (N = 5/group), **P = 0.0027. G-I In the absence (left bars) and presence (right bars) of Hyp + SU5416 for 2 weeks, siISCU (black bars) induced elevations in RVSP (***P < 0.0001, **P = 0.0031) (G) and increased pulmonary vascular remodeling in < 100-μm pulmonary vessels (H, I) as compared with siCont (N = 5/group, ***P = 0.0003, *P = 0.0114 for H; ***P = 0.0006, **P = 0.0098 for I). G-I In the absence (left bars) and presence (right bars) of Hyp + SU5416 for 2 weeks, siISCU (black bars) induced elevations in RVSP (***P < 0.0001, **P = 0.0031) (G) and increased lung remodeling in < 100-μm pulmonary vessels (H, I) as compared with siCont (N = 5/group, ***P = 0.0003, *P = 0.0114 for H; ***P = 0.0006, **P = 0.0098 for I). J Model of acquired and genetic Fe-S deficiency as a central cause of metabolic dysfunction and PH in mice and humans. Among others, potential translational implications are highlighted in red text.
Detection of ctDNA in plasma, urine and CSF is affected by intra-tumor heterogeneity in gliomas.Figure 3A: Mutant allele fraction (MAF) measured in tumor, CSF, plasma and urine samples (on the y axis) for the detected mutations, in the 52 genes that are most frequently mutated in gliomas based on the TCGA databases (on the x axis), for the 8 patients analysed by targeted sequencing panels. MAF from tumor are derived from exome sequencing. MAF from CSF, plasma and urine samples are derived from the capture panels sequencing. B: Number of mutations detected in tumor samples that were observed across several individual tumor biopsies (shared) or private to an individual biopsy. C, D, E: Comparison of the number of mutations that were detected in multiple tumor tissue subparts (red), that were private to an individual subpart (blue), or that were rescued by merged calling (green) in CSF (C), plasma (D), and urine (E). Data are shown for samples collected immediately prior to surgery across all 8 patients.
Figure 4Schematic of the experiment. K and KM animals were set on doxycycline food until mammary tumours developed. Primary tumours were collected and single cells injected into Rag2-/- animals to recapitulate the tumours followed by either no treatment, drug treatment or switched to a normal diet. Relative volume of tumours grown in 12 Rag2-/- animals after treatment with Tepotinib or vehicle control (3K and 3KM tumours for each condition). No statistical significance was found by One-way ANOVA. Mean +/- SEM. Quantification of cMet copy number detected by digital PCR or FISH in primary tumours injected into Rag2-/- and treated with vehicle control or Tepotinib. Quantification of cMet copy number detected by digital PCR or FISH in primary tumours injected into Rag2-/- and fed with doxycycline or after doxycycline withdrawal.
Fig.Figure 5δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (A) Mice were monitored for weight loss at indicated time-points. δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (B) IAV titre in lungs was determined by plaque assay at 5 d.p.i. (n = 6). δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (C) IL-1β mRNA transcripts in lung at 5 d.p.i. were determined by qPCR. δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (D) Precision-cut lung slices from control and δWD mice were infected with IAV. Virus titres were determined at indicated time points.
Energy metabolism of mitochondria.Figure 4(A-F) OXPHOS complexes (A-C) and selected enzymes involved in the energy metabolism of control (143B, solid lines) and ρ0 cells (dashed lines). A, Complex (C) I, III and IV; B, F1, FO domain and inhibitor protein IF1 of complex V; C, subunits SDHA, SDHB-D and assembly factor SDHAF2 of complex II; D, oxoglutarate (OGDH; average of OGDHL, OGDH, DLST) and pyruvate dehydrogenase complex (PDH; average of PDHA1, PDHB, PDHX, DLAT); E, subunits of GDP forming (SUCLG2) and ADP forming (SUCLA2) succinyl-CoA ligase; F, urea cycle enzymes and glutaminase (GLS). Plots and gene names are in corresponding colors.
a) Subcellular fractionation and immunoblotting.f7(a) Subcellular fractionation and immunoblotting. Jurkat T cells were cultured in the presence of etoposide for the indicated times. Following etoposide treatment, the ATG5 monomer was newly present in both cytoplasm and nucleus. The exposure of the ATG5 monomer (shown under the dotted line) was longer than that shown above for the ATG5 conjugated to ATG12. Full-length immunoblots are provided in Supplementary Fig. S10. b) A comparison of ATG5 sequences in different species demonstrates the presence of a conserved putative NES, similar to that present in Beclin 1. The sequence alignment was performed with Clustal W using sequence data obtained from GenBank. The characteristic leucines are indicated in red and the resulting sequence of ATG5-ΔNES, following mutation of three leucines within this motif, is shown. (c) (c) Subcellular fractionation and immunoblotting of the nuclear fraction. Jurkat T cells, either treated with etoposide, or overexpressing ATG5 following lentiviral gene transfer, were cultured in the presence and absence of leptomycin B for 72 h. Leptomycin B increased relative ATG5 and Beclin 1 levels in the nucleus. (d) Subcellular fractionation and immunoblotting. Jurkat T cells were transduced to express ATG5-ΔNES, ATG5, or GFP. Although increased levels of ATG5 were observed in both cytoplasm and nucleus, ATG5-ΔNES accumulated following lentiviral gene transfer in the cytoplasm only. Full-length immunoblots are provided in Supplementary Fig. S11. (e) Immunoblotting. ATG5-ΔNES overexpression induced LC3-II formation and p62 degradation, indicating increased autophagic flux. (f) Cell cycle analysis. Jurkat T cells overexpressing ATG5 or ATG5-ΔNES, as well as control cells were cultured for 48 h. Induction of G2 and M phase arrest was seen as a consequence of ATG5, but not ATG5-ΔNES overexpression. As an additional control, we transduced cells with GFP, which, similar to ATG5-ΔNES-overexpressing cells, failed to exhibit G2- and M-phase arrest. Representative flow cytometry diagrams are shown (n=3). (g) Morphological analysis. Non-transduced Jurkat T cells and cells overexpressing ATG5 or ATG5-ΔNES were cultured for 48 h. Large, multinucleated cells were seen as a consequence of ATG5, but not ATG5-ΔNES overexpression. Representative examples are shown (n=3). Scale bar, 10 μm.
Phb2pko mice develop glomerulosclerosis.Figure 2Phb2pko mice develop glomerulosclerosisA PAS staining of kidney sections (scale bar: 20 μm).B Immunohistochemistry for podocin on kidney sections (scale bar: 20 μm).C Analysis of podocyte foot processes in electron micrographs (scale bar: 0.7 μm). FP, podocyte foot processes; GBM, glomerular basement membrane; EC, endothelial cells.D Analysis of mitochondrial morphology in electron micrographs (arrows; scale bar: 0.3 μm).E High-power view of mitochondria in electron micrographs (scale bar: 0.1 μm).
GFP–Atg8F77/79AΔR double mutant shows a defect in the autophagic process despite undergoing lipidation.Figure 4(A) Cell extracts were subjected to 13.5% SDS-polyacrylamide gel electrophoresis containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐15N peptide antibodies. (B) Cells were grown in control or in starvation medium and visualized using fluorescence microscopy. Arrows point to the pre‐autophagosomal structure, whereas arrowheads point to GFP within the vacuoles. (C) Aminopeptidase I (API) maturation and GFP cleavage were tested as described above. Asterisk represents nonspecific band. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.