EMBO/sd-character-level-ner huggingface dataset:
Figure 1A. BrdU-sensitized U-2-OS Flp-In T-REx cells stably expressing mCherry-FAN1 and GFP-NLS or GFP-CDKL5 (no NLS) were line micro-irradiated (355 nm) and imaged after 2 min. Scale bar is 10 μm B. BrdU-sensitized U-2-OS cells stably expressing GFP-NLS-CDKL5 were pre-incubated with DMSO (mock), olaparib (5 μM), talazoparib (50 nM) or PDD00017273 (0.3 μM) for 1 h prior to micro-irradiation and live-imaging at the indicated times post-irradiation. One of three independent experiments is shown. Scale bar is 10 μm C. Same as B. except that cells stably expressing GFP-NLS-CDKL5 were pre-incubated with DMSO (mock), olaparib (5 μM) or PDD00017273 (0.3 μM) for 1 h prior to spot micro-irradiation (405 nm). Individual cells from one of two independent biological replicates is shown. Scale bar is 10 μm D. Quantitation of spot intensities. Data represent the mean ± SEM of two independent experiments; > 50 micro-irradiated cells per point. E. BrdU-sensitized parental or PARP1Δ/Δ, PARP2Δ/Δ, PARP1/2Δ/Δ U-2-OS cells transiently expressing GFP NLS-CDKL5 were subjected to 355 nm line micro-irradiation followed by time lapse imaging. One of two independent experiments is shown. Scale bar is 10 μm F. Diagram of the workflow for the chromatin retention experiments. G. Cells subjected to the workflow in F. were detergent-extracted and fixed before staining with anti-GFP or fibrillarin (nucleoli). Scale bar is 10 μm H. Quantification of the detergent-insoluble GFP-NLS-CDKL5 signal (minus nucleolar signal). The mean ± SD from three biological experiments is shown. Statistical significance was assessed by one-way-ANOVA-test. Asterisks ** indicate P-value of <0.01; ns - not significant.
Fig.Figure 2A Heat map analysis of 17 sterol biosynthesis genes and 3 other SREBP2 target genes from RNA-seq data using p53+/+ and p53-/- HCT116 cells. Expression levels were normalized to the mean level of each gene among all samples and compared to p53+/+ cells. Color scale indicates the expression fold change of target gene. B mRNA expression of SQLE in the mouse liver of p53+/+ and p53-/- mice with normal or HFD diet. C mRNA and protein levels of SQLE in different tissues of p53+/+ or p53-/- mice were determined by qRT-PCR and western blot respectively. SQLE protein expression and mRNA levels were examined in p53+/+ and p53-/-HCT116 cells (D) Actin was used as loading control. E SQLE protein expression and mRNA levels were examined in p53+/+ and p53-/-HCT116 HepG2 cells (E). Actin was used as loading control. F and G p53+/+ and p53-/- HepG2 cells were cultured for 48 hours in medium containing fetal bovine serum (Serum) or lipoprotein-depleted FBS (LPDS). Protein expression was shown by western blotting (F). mRNA levels of SQLE were examined by qRT-PCR (G). P: premature SREBP2; M: mature SREBP2. Protein expression and mRNA levels of p53+/+ and p53-/- HepG2 cells treated with control siRNA or three separated sets of SREBP2 siRNAs for 72 hours as indicated. I and J Protein expression and mRNA levels of p53+/+ and p53-/- HepG2 cells treated with control siRNA or three separated sets of SREBP2 siRNAs for 72 hours as indicated. K p53+/+ and p53-/- HepG2 cells were treated with control siRNA or SREBP2 siRNA, followed by ectopic expressed RNA-resistant SREBP2 for 48 hours. mRNA and protein expression were examined by qRT-PCR and western blotting.
A) FACS analysis of TUNEL positive p27+/+ and p27−/− cells treated with AICAR for 20 h.figf5(A) FACS analysis of TUNEL positive p27+/+ and p27−/− cells treated with AICAR for 20 h. Data represent mean ± s.d. of three independent experiments. (B, C) Electron microscopy of p27+/+ (a, c and e) and p27−/− (b, d and f) MEFs treated with (c and d), or without (a and b) AICAR (1 mM), or without glucose (e and f) for 14 h. Magnified images of the boxed areas were shown on the left (a′, b′, c′, d′, e′ and f′) with arrowheads indicating autophagic vacuoles including autolysosomes (black arrowheads). The scale bars represent 10 μm in a-f and 0.5 μm in a′-f′. N, nucleus. Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s.d. (C). (B, C) Electron microscopy of p27+/+ (a, c and e) and p27−/− (b, d and f) MEFs treated with (c and d), or without (a and b) AICAR (1 mM), or without glucose (e and f) for 14 h. Magnified images of the boxed areas were shown on the left (a′, b′, c′, d′, e′ and f′) with arrowheads indicating autophagic vacuoles including autolysosomes (black arrowheads). The scale bars represent 10 μm in a-f and 0.5 μm in a′-f′. N, nucleus. Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s.d. (C).
Reduced immune response of myeloid cells to LCMV infection under GF conditions.Figure 6(A) Iba1 immunohistochemistry depicting cortical microglia, meningeal macrophages, perivascular macrophages and choroid plexus macrophages 4 days after i.c. challenge with LCMV or vehicle controls. Arrows indicate Iba1+ meningeal or perivascular macrophages, respectively. (B) Quantification of Iba1+ microglia, choroid plexus macrophages, meningeal macrophages and perivascular macrophages. Each symbol represents one mouse. Three to four sections per mouse were examined. Significant differences were evaluated by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments. (C, D) Representative immunofluorescence images (left) of (C) Iba1 (green) and KI67 (red) or (D) CD74 (red) on cortical sections from SPF and GF mice. Nuclei were stained with DAPI (blue). Scale bars: 25 μm. White arrows indicate KI67+/Iba1+ or CD74+/Iba1+ macrophages and arrowheads show KI67-/Iba1+ or CD74-/Iba1+ macrophages. Quantification thereof (right). Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were evaluated by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.
Overall structure of 2019-nCoV RBD bound with ACE2.Fig. 1Overall topology of 2019-nCoV spike monomer. NTD, N-terminal domain. RBD, receptor-binding domain. RBM, receptor-binding motif. SD1, subdomain 1. SD2, subdomain 2. FP, fusion peptide. HR1, heptad repeat 1. HR2, heptad repeat 2. TM, transmembrane region. IC, intracellular domain. Sequence and secondary structures of 2019-nCoV RBD. The RBM is colored red. Overall structure of 2019-nCoV RBD bound with ACE2. ACE2 is colored green. 2019-nCoV RBD core is colored cyan and RBM is colored red. Disulfide bonds in the 2019-nCoV RBD are shown as stick and indicated by yellow arrows. The N-terminal helix of ACE2 responsible for binding is labeled.
Figure 1a, Cells (wt, ∆atg18 or ∆atg18/∆atg21/∆hsv2 ()), with and without a plasmid expressing wt or a mutant form of Atg18) were grown in YPD, vacuoles were stained with FM4-64 and imaged by confocal microscopy. At least 10 confocal sections spaced at 300 nm were assembled into maximum projections. Images show cells before (0 min) and 15, 30 and 60 min after induction of vacuolar fragmentation by addition of 0.4 M NaCl. Red: FM4-64. Gray: DIC. Scale bar 5 µm. b, The number of vacuoles per cell was quantified for the experiment in a. 150 cells from three independent experiments were counted for each condition. Values represent the mean, error bars the SEM.
Dissection of the energetic contributions to GK efficiency.Figure 4A) Left panel: overlay of the models resulting from the introduction of Arg and Asp into the 2M5N zipper structure, with the identifier indicated for each strand. Strands E and F are highlighted in red. Right panel: the same structure turned 90° about both the x- and y-axes. Arrows in (a) indicate the capacities of Arg and Asp to distance their charged moieties. (b) shows an example of planar Arg stacking. B) Boxplots showing all-atom analysis of the energetic effects of the introduction of GKs into a set of amyloid steric zipper structures. The upper whiskers indicate the largest value no further than 1.5 times the Inter-Quartile Range (IQR) from the upper hinge, the lower whiskers show the smallest value at least 1.5 * IQR from the lower hinge. Notches extend 1.58 * IQR / sqrt(n) from either side of the median and represent a 95% confidence interval for the median value. Grey shaded area indicates total energy associated with the introduction of GKs into zipper structures, the remainder of the plot shows the breakdown of this total energy in into constituent energy terms. Each boxplot shows the distribution of 72 observations. C) Violin plots showing the distribution of distances to the closest charged moiety in a neighboring GK residue in the central-most positions (strands E and F) of the steric zipper models. Each violin shows the distribution of 83 observations. D) Time-resolved Dynamic Light Scattering (DLS) analysis showing average particle radius over time for the β-Gal peptides flanked by both natural and non-natural GKs, at increasing NaCl concentrations (indicated in µM). Structures of the amino acids flanking the aggregating core are indicated on the right-hand side, the sequence of the aggregating core is indicated below.
Facilitated OGS promotes reprogramming (A-B) 5C or mES medium was used during reprogramming.Figure 15C or mES medium was used during reprogramming. On day 15, Oct4GFP+ colonies were counted (A). 5C or mES medium was used during reprogramming. The percentages of Oct4GFP+ cells were also determined with FACS (B). (C) The expression of several core pluripotency markers was determined with qPCR at different time points during reprogramming and normalized against those in MEFs. (D-E) Energy metabolism was analyzed on day 6 during reprogramming with the Seahorse instrument. Increase in extracellular acidification rate (ECAR) after adding glucose was considered as glycolysis ability of the cells (D), while the decrease in oxygen consumption rate (OCR) was considered as the ATP production ability of the cells (E). (F-G) Expression of Hif1α was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming (F). The numbers of Oct4GFP+ colonies were determined on day 15 (G). (H-I) Expression of Pdk1/2 was modulated with a retrovirus system, and oligomycin (1 µM) and 2-DG (5 mM) were used during reprogramming (H). The numbers of Oct4GFP+ colonies were determined on day 15 (I).
Figure 2A-D Microtubule affinity measured by a cosedimentation assay in the apo state (full line) and in the presence of ATP (dashed line), and AMPPNP (dotted line) for wild-type (A), mutant 2 (B), mutant 5 (C), and mutant 13 (D). E. Microtubule stimulated ATPase activity of wild-type (grey), mutant 2 (red), mutant 5 (orange), and mutant 13 (purple). F. Bar plot of basal ATPase activity of wild-type (grey), mutant 2 (red), mutant 5 (orange), and mutant 13 (purple). Data information: Error bars show standard deviation of three repetitions of different dynein preparations.
Tyrosine phosphorylation reduces contacts and aggregation of disease mutants.Figure 4A) After cleavage of a C-terminal maltose binding protein solubility tag, hnRNPA2 FL WT undergoes LLPS and disease mutants D290V and P298L aggregate. In contrast, phosphorylated hnRNPA2 FL WT and P298L do not undergo LLPS or aggregation in the time frame tested. Phosphorylated hnRNPA2 FL D290V can form some structures resembling liquid droplets but does not form amorphous aggregates in the time frame tested. Conditions: 20 µM proteins, 20 mM Tris pH 7.4, 50 mM NaCl. Scale bar: 10 µm B) PLAAC analysis (Lancaster et al., 2014) of hnRNPA2 (black), changing all tyrosine residues in hnRNPA2 LC to glutamate (17 YE, red), 5 serine to glutamate phosphomimic mutations (5 SE, blue), all serine (12 SE, purple), and all tyrosine to glutamine (17 YQ) shows that hnRNPA2 LC prion-like character reduces slightly with serine phosphomimic mutations, but decreases drastically with tyrosine phosphomimic mutations. C) As the number of phosphorylation events increases, the radius of gyration (Rg) of hnRNPA2 LC increases in coarse-grained simulations. Rg uncertainty bars represent S.E.M. from 10 different simulations with the same number of phosphorylated tyrosines randomly placed in different positions. D) Coarse grained simulations of WT show that compared to the unphosphorylated form, phosphotyrosine hnRNPA2 LC forms fewer contacts with itself.
Basal levels of RARα, RARβ, and RARγ mRNA splicing variants in mammary tumors.Figure 7Basal levels of RARα, RARβ, and RARγ mRNA splicing variants in mammary tumorsTotal RNA was extracted from the tissue slices deriving from the surgical specimens of breast cancer patients used in Fig3 before any treatment with DMSO or ATRA. RNA was subjected to RT-PCR analysis to determine the basal expression of the indicated RAR splicing variants.Each value represents the mean ± SD of two replicate measurements. The table shows the statistical significance of the indicated comparisons. *Significantly different (P-value < 0.05, Student's t-test). **Significantly different (P-value < 0.01, Student's t-test). The plots illustrate the average expression levels of the indicated mRNAs (mean ± SD of two replicates) in tumor samples classified as ATRA-sensitive (Sens) and ATRA-resistant (Res) according to the response of Ki67. *Significantly different (P-value < 0.05, Student's t-test).
Figure 2A. Immunoblot analysis of cleaved GSDMD in culture supernatants and full length GSDMD, cleaved GSDMD and α-tubulin in the cell lysates of immortalized LPS-primed WT macrophages left uninfected (NS) or infected for 10-40 minutes with log phase S. typhimurium (MOI=50). B. Schematic representation of the subcellular fractionation shown in C. C. Fractionation and immunoblot analysis for GSDMD, Na+K+ ATPase, VDAC (Voltage-dependent anion channel), HDAC1 (Histone deacetylase 1) and GAPDH (Glycerinaldehyd-3-phosphat-Dehydrogenase) of WT macrophages infected for 10 and 20 minutes with log phase S. typhimurium (MOI=50). Fractionation was carried out as described in the Material and Methods section and equivalent amount of protein was loaded per lane. D. Extraction of cleaved GSDMD from isolated membranes of WT macrophages infected for 10 minutes with log phase S. typhimurium (MOI=50). Extraction was carried in variable conditions as described in the Material and Methods section.
a) Schematic representation of full-length UVRAG with the boundary of the C2 domain indicated above the diagram.figf1b) The affinity of the UVRAG-C2 domain for phospholipids was assessed using a protein-lipid overlay assay. The left panel indicates the identity of lipid species on PtdIns strips. Bacterial-purified GST fusion of UVRAG-C2 (wild type, WT), but not GST, binds PtdIns(3) P, PtdIns(4) P and PtdIns(5) P. The K78A/R82A mutant of C2 is defective for phospholipid-binding, whereas the K87A/N88A mutant is partially impaired. Binding of the FYVE and PH domains of Hrs and FAPP1, respectively, to PtdIns(3) P and PtdIns(4) P served as quality controls (right). PE, phosphatidylethanolamine; PC, phosphatidylcholine; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; S1P, sphingosine 1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine. (c) The relative affinity of the GST-fusion proteins for phosphoinositides was quantified by densitometric analysis of the GST immunoblots in b from five independent experiments (n = 5). Error bars represent standard deviation (s.d.). A.U., arbitrary units. (d) Binding of full-length UVRAG (untagged) to lipid blots. The same PtdIns strips (in b) were used and immunoblotted with antibodies against IgG (right) and UVRAG (left). (g) Pulldown of UVRAG-C2 domain GST-fusion proteins by liposomes bearing the indicated phosphoinositides. GST-Hrs-FYVE and GST-FAPP1-PH are also shown.
TGIF1 associates and with and inhibits Twist1 expression A-C Protein extracts from MIAPaCa-2 cells (left) and control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 (A-C).Figure 3Protein extracts from MIAPaCa-2 cells (left) and control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 (A-C). Protein extracts from control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 To control for loading, total cell lysates were analyzed by immunoblotting using antibodies to Twist1, TGIF1 or β-Actin. Protein extracts from MIAPaCa-2 cells (left) and control were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 In C, cell lysates were treated with DNase I before coimmunopreciptation. Panc-1 cells were treated with TGF-β for 30 min, and protein extracts were immunoprecipitated with anti-TGIF1 antibody and analyzed by immunoblotting using anti-Twist1 antibody. To control for activation of TGF-β signaling, total cell lysates were analyzed by immunoblotting using anti-pSmad2 antibody. PL45 stably expressing Dox-inducible control or sh-RNA targeting TGIF were treated with vehicle or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6). In left panel, the intensity of the bands was assessed by densitometry, and the result was presented as a ratio of Twist1/TGIF1. PL45 stably expressing Dox-inducible TGIF1 were treated with vehicle or Dox for 48 hr and analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel. Pancreatic chromatin from control or Tgif1KO mice was analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.
Figure 5(A) WT, Irgm2-/-, and Nlrp3-/- BMMs were treated with LPS (0.1 µg/ml) for 3 h followed by nigericin for 1 h and IL-1β/LDH release was measured. (B) WT, Irgm2-/-, Casp11-/- and Irgm2-/-Casp11-/- BMMs were treated with LPS (0.1 µg/ml) for 3 h followed by nigericin and/or MCC950 for 1 h and IL-1β/LDH release was measured. (C) WT, Irgm2-/-, Casp11-/- and Irgm2-/-Casp11-/- BMMs were treated with LPS (1 µg/ml) and/or MCC950 for 24 h and IL-1β/LDH release was measured. (D) IFNγ-primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were transfected with LPS using lipofectamine LTX and LDH release was measured at 2 hpt. (E) IFNγ-primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were co-treated with LPS (indicated doses) and Listeria monocytogenes (MOI 10) and LDH release measured at 4 hpt.
Figure 5Left image: SUD generally stimulates protein translation level detected by the luciferase-pcDNA3 reporter (n=6). Middle image: SUD does not increase the amounts of total protein synthesis (host and viral proteins) in the replicon-transfected cells. Right image: SUD augments viral protein synthesis (n=8). HEK-293 cells growing in 12-well plates were transfected with the indicated plasmids and replicon DNA. Twenty-four hours post-transfection, cells were harvested for Renilla luciferase activity measurement (left and right image). For the ribopuromycylation assay, 24 hours post-transfection cells were pulsed with 3 µM puromycin for 1 hour at 37°C before harvesting for western blot analysis (middle image). Δ16-SUD protein is not detectable but the mRNA level is normal. HEK-293 cells were transfected with the indicated plasmids. Twenty-four hours post-transfection, cells were fixed for DAPI staining (left image) or lysed for a SYBR Green q-PCR assay (right image, n=6). Scale bars represent 100 µM. Left image: Mac2 stimulates replication of pBAC-ΔMac2-REP-Rluc replicon, but Δ16-Mac2 does not (n=6). Right image: Cellular RNA was isolated for qPCR assay using primers and probes specifically recognizing SARS Nsp14 and ß-actin. The relative pBAC-REP-Rluc-related replicons RNA level was calculated as the ratio of Nsp14 RNA to ß-actin RNA. n=5.
THEMIS knock‐down increases TCR signalling in peripheral CD4+ T cellsLentiviral knock‐down of THEMIS expression in primary human CD4+ T cells.Figure 4A Lentiviral knock‐down of THEMIS expression in primary humanCD4+T cells. Following transduction, cells were expanded on CD3/CD28 beads in the presence of exogenous IL‐2 and analysed for THEMIS KD by immunoblotting on day 4 post‐transduction. Data shown are representative of three independent experiments. B pERK response in THEMIS KD humanCD4+T cells. Transduced CD4+ cells were removed from beads, rested overnight and stimulated with CD3 mAb for the indicated time points. ERK phosphorylation was assessed by immunoblotting. Data shown are representative of three independent experiments. C Expression of CD25 and CD69 surface markers of THEMIS KD humanCD4+T cells was assessed on day 4 post‐transduction by flow cytometry. Data shown are representative of three independent experiments. D CD69 up‐regulation on re‐stimulated THEMIS KD CD4+T cells. CD3/CD28 beads were removed on day 1 post‐transduction, and cells were cultured in IL‐2 and IL‐7. After removal of cell debris at day 4, cells were restimulated with CD3/CD28 beads at the indicated bead to cell ratio. CD69 surface expression was assessed by flow cytometry 24 h later. Data shown are representative of two independent experiments. n = 3, means ± SD are shown; two‐tailed unpaired Student's t‐test, *P 0.05, ***P 0.001.
Figure 6A HEK293 cells were transfected with increasing amounts of Flag-TRIM33 expression vectors and 48 hours after transfection, cell lysates were subjected to immunoblotting (IB) using antibodies as indicated. B HEK293 cells were transfected with increasing amounts of Flag-FBXW7 expression vectors and 48 hours after transfection, cell lysates were subjected to immunoblotting (IB). C HEK293T cells were transfected as indicated, at 18 h after transfection, the cells were treated with the proteasome inhibitor MG132 (20μM) for 6 hours and then harvested. Cell lysates were immunoprecipitated by Flag-antibody (M2 beads) and Ubiquitin-conjugated GPX1 was detected by Western blotting with anti-Myc antibody. D HEK293 cells were transfected with either control or CUEDC2 siRNA and increasing amounts of Flag-TRIM33 expression vectors, cell lysates were subjected to immunoblotting (IB) using antibodies as indicated.
Figure 8A Immunoblotting for CUEDC2 protein level. 4 weeks after tail-vein injection of rAAV9-CUEDC2, indicated mice tissues were harvested and subjected to immunoblotting to detect CUEDC2 protein level. Ctl: heart of rAAV9-GFP injected mice. B Animals were injected by rAAV9-CUEDC2 in a different amount of viral genomes (VG). Heart tissues were then analyzed by immunoblotting to detect CUEDC2 transduction. Tubulin protein level was examined for normalization purposes. C The ratios of AAR to LV, IRI to AAR and IRI to LV are shown. n = 10 mice in WT + rAAV-GFP group and Cuedc2-/- + rAAV-GFP group; n = 12 mice in Cuedc2-/- + rAAV-CUEDC2 (1×1010 VG) group and Cuedc2-/- + rAAV-CUEDC2 (1×1011 VG) group. * p = 0.0055, ** p = 0.0146, *** p = 0.0200, **** p = 0.0377. D Effect of rAAV9-CUEDC2 treatment on echocardiography indices of left ventricle function recovery post-I/R measured by fractional shortening (FS) and ejection fraction (EF). WT+ rAAV-GFP (n = 10); Cuedc2-/- + rAAV-GFP (n = 10); Cuedc2-/- + rAAV-CUEDC2 (Low dose) (n = 12); Cuedc2-/- + rAAV-CUEDC2 (High dose) (n = 12). ** p < 0.0001.