EMBO/sd-character-level-ner huggingface dataset:
Figure 2(A) HeLa cells were transfected with the indicated siRNAs. After 72 h, the cells were subjected to immunofluorescence assay using antibody against mTOR (red). DAPI (blue) was used for nuclear staining. The stained cells were visualized using confocal microscopy. Scale bar, 5 µm. (B) HeLa cells were transfected as in (A). After 72 h, cells lysates were subjected to immunoblot analysis using anti-TDP-43, p70S6K, phosphorylated p70S6K (T389), mTOR, RagB, P18, raptor and GAPDH antibodies. The relative densities of phosphorylated/total p70S6K are shown on upper-right panel, and the relative densities of mTOR, RagB, P18, raptor and TDP-43 to GAPDH are shown on lower-right panel). The data from three independent experiments are presented as means ± S.E.M., ns, not significantly different; *, p<0.05; **, p<0.01, one-way ANOVA. (C) HeLa cells were transfected with the indicated siRNAs. After 48 h, the cells were re-transfected with LAMP1-RFP (red) for 24 h. The cells were then incubated with Earle's Balanced Salt Solution (Starvation) for 2 h or restimulated with amino acids (starvation + amino acids) for 30 min. The cells were stained with mTOR (green) and DAPI (blue), and then visualized using confocal microscopy. Regions within the dotted boxes are magnified in the insets. Scale bar, 5 μm. (D) Similar transfection and treatment as in (C) were performed and the lysates from starved and re-feeding cells were subjected to immunoblot analysis using anti-TDP-43, p70S6K, phosphorylated p70S6K and GAPDH antibodies. The relative densities are shown on the right side. The data from three independent experiments are presented as means ± S.E.M., ns, not significantly different; **, p<0.01, one-way ANOVA. (E) Similar transfection as in (B) was performed, and transfected cells were processed for qRT-PCR analysis. The level of TDP-43, mTOR, raptor, RagB and P18 mRNA were quantified and normalized relative to GAPDH. The data from three independent experiments are presented as means ± S.E.M., ns, not significantly different; **, p<0.01, one-way ANOVA. (F, G) HeLa cells were transfected with the indicated siRNAs. After 48 h, the cells were transfected with LAMP1-RFP (red), with or without HA-raptor for 24 h. The cells were stained with anti-mTOR (green) antibody and DAPI (blue), and then visualized using confocal microscopy. Regions within the dotted boxes are magnified in the insets. Scale bar, 5μm. The quantification of mTOR on lysosomes is shown in Figure 2G. Data from three independent experiments represented as means ± S.E.M., **, p<0.01, one-way ANOVA.
Figure 1Western blotting analysis of Gadd45a protein expression in mouse intestine crypt lysates from WT and G3Terc-/- mice (A). Quantification of Gadd45a protein expression, β-actin protein level was used as the loading control (n=7 mice per genotype), two independent experiments were carried out (B). Quantification of Gadd45a-ChIP values as percentage of total telomeric DNA and ALU elements in WT and G3Terc-/- MEF cells (n=3 for each genotype). Dot-bot assay following Gadd45a-ChIP in WT and G3Terc-/- MEF cells. Kaplan-Meier survival curves of the mice with indicated genotypes (WT, n=44; Gadd45a-/-, n=48; G3Terc-/-, n=37; G3-dKO, n=41). P value comparing the survival of G3Terc-/- with G3-dKO mice is calculated with the log-rank test. The average body weight of mice with indicated genotypes at indicated time points (G3-dKO Female (n=19) vs G3Terc-/- Female (n=17), P=0.004; G3-dKO Male (n=14) vs G3Terc-/- Male (n=14), P=0.0038). P values are calculated with log-rank test. Whole mount images of colon samples from 7-month old mice with indicated genotypes (G). Quantification on density of colon crypts (H) (n=5 mice per group). H&E-stained small intestine sections from 7-month old mice with indicated genotypes (I). Quantification of the number of small intestine basal crypts (J) (n=5-6 mice per group).
a–e, Scanning electron microscopy (SEM) images of fly eyes expressing DTS7 with or without the indicated HDAC6 transgenes.figf1a-e, Scanning electron microscopy (SEM) images of fly eyes expressing DTS7 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. a, Normal eyes in DTS7 flies reared at 22 °C. b, Rough eyes in DTS7 flies reared at 28 °C. c, d, Degeneration was suppressed by expression of Drosophila HDAC6 (dHDAC6; c) or human HDAC6 (hHDAC6; d), but not a catalytically dead mutant of hHDAC6 (cat. mut.; e). f-j, SEM images of fly eyes expressing AR52 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. f, Normal eyes in AR52 flies reared without (-) DHT. g, Rough eyes in AR52 flies reared with (+) DHT. Degeneration was suppressed by expression of Drosophila HDAC6 (h) or human HDAC6 (i), but not a catalytically dead mutant of human HDAC6 (j). k-p, Detection of UPS reporter in imaginal eye discs from third-instar larvae by confocal microscopy. High level fluorescence was found in flies expressing GFP (k, positive control), but fluorescence was barely detectable in control flies expressing CL1-GFP (l, negative control). CL1-GFP accumulates in DTS7flies with temperature-dependent proteasome impairment (compare m to n) and in AR52flies with ligand-dependent degeneration (compare o to p). The retinal phenotypes of 200 to >1,000 flies of each genotype were examined. Quantitative analyses of eye phenotypes and proteasome impairment are presented in Supplementary Figs S2 and S3, respectively. (DHT, dihydrotestosterone.)
The loss of ATG13 causes a shift in colocalization of ATG9A with organelle and autophagy markers and triggers an accumulation of ATG9A with p62/SQSTM1.Figure 3(A)-(G) Representative images of ATG9A colocalization with different organelle and autophagy markers. HCT-116 ATG9A-HA KI-ATG13 WT or ATG13 KO cells were grown in full DMEM media, fixed and labeled with antibodies for HA and indicated organelle/cellular markers and imaged (Scaler bar=10 µm). (H) Quantification of ATG9A colocalization with indicated organelle markers of golgi (GOLGIN97), mitochondria (TUFM), ER (PDIA3), endosomal system (VPS26A), early endosome (EEA1), lysosome (LAMP1), autophagy adaptor (p62), and autophagosome markers (ATG16L1, WIPI4, ATG2A, LC3II, WIPI2) respectively. Mean ± SEM, n=1 independent experiment with 30 technical replicates. Images for p62 colocalization (G) are from the same single independent experiment
Figure 3A. HME cells expressing wild-type, T387A, or T387D STAT2 were seeded in 96-well plates at 8000 cells/well. The cells were exposed to vesicular stomatitis virus (VSV) for 2 h, with or without pre-treatment with IFN-β (100 IU/ml). After 20 h, the amounts of the VSV-M and STAT2 proteins were analyzed by the Western method. B, C. HME cells expressing wild-type, T387A, or T387D STAT2 were seeded in 96-well plates at 8000 cells/well. On the second day, the cells were treated with IFN-β (10 IU/ml) for 2 h. The cells were then exposed to different multiplicities of infection of VSV for 2 h, followed by fresh media containing 10 IU/ml of IFN-β. The cells were analyzed by staining with crystal violet (B) after 48 h. B, C. HME cells expressing wild-type, T387A, or T387D STAT2 were seeded in 96-well plates at 8000 cells/well. On the second day, the cells were treated with IFN-β (10 IU/ml) for 2 h. The cells were then exposed to different multiplicities of infection of VSV for 2 h, followed by fresh media containing 10 IU/ml of IFN-β. The cells were analyzed by the MTT assay (C) after 48 h. D. U6A cells expressing wild-type, T387A, or T387D STAT2 were placed into 96 well plates (2000 cells/well) and the cells were treated with IFN-β (1000 IU/ml) for 96 h. Cell survival was analyzed by the MTT assay. Each experiment was carried out two independent times, with results similar to the representative examples that are shown.
Image-guided microinjection protocol and model system: A.Figure 2Schematic of targeted telencephalon: Left, E14.5 brain; middle, coronal section of the brain; right, inset illustrating the organization and major cell types of the cortical wall. APs attached to the ventricle are targeted by automated microinjection. Microscope images of the tissue and microinjection pipette before (top) and after (bottom) annotation of the tissue surface and microinjection micropipette tip by the experimenter. Scalebars: top 100µm, bottom 10µm. Ap indicates the apical surface and BL indicates the basal lamina. A cartoon schematic of the microinjection protocol. (i) The user brings the micropipette into the microscope FOV close to the tissue. (ii) The manipulator is calibrated to allow for image-guided positioning of the micropipette using a calibration algorithm (see Appendix Note S1, and Appendix Figure S2 for additional details). (iii) The user annotates image with the desired trajectory of microinjection by dragging a cursor over the edge of the tissue, and clicks a point indicating the tip of the micropipette. (iv) Before injection begins, the user brings the tip of the micropipette close to the tissue surface and specifies the starting point. (v) The following steps are fully automated. The Autoinjector positions the micropipette at the surface of the tissue and advances the micropipette to a specified depth under pressure into the tissue resulting in microinjection. The micropipette is then retracted out of the tissue and repositioned at the next microinjection site. This is repeated until the micropipette reaches the end of the microinjection trajectory. The user can independently specify the depth of microinjection, retraction, and spacing between microinjections (see Appendix Note S2, and Appendix Figure S2 for additional information). Ap indicates the apical surface and BL indicates the basal lamina.
cGAMP-VLPs containing the SARS-CoV-2 S protein induce an enhanced antibody response.Figure 7A. cGAMP-S-VLPs and Empty-S-VLPs incorporate SARS-CoV-2 S. Lysates from VLP producer cells and VLP preparations were analysed by western blot for the presence of the S2 subunit of SARS-CoV-2 S, Gag-GFP and actin using the indicated antibodies. B, C. Immunisation with cGAMP-S-VLPs augments anti-Spike and anti-RBD antibody titres. Mice were immunised via the intra-muscular route with 5x105 IU of cGAMP-S-VLPs, Empty-S-VLPs, or PBS as a control. Three weeks after immunisation, sera were collected, heat-inactivated, and titres of antibodies capable of binding to SARS-CoV-2 S (B) or its RBD (C) were determined by ELISA. The antibody response was expressed as Endpoint titre defined as the reciprocal of the highest serum dilution that gives a positive signal (blank+10SD). The dotted line shows the limit of detection (LOD). D. Immunisation with cGAMP-S-VLPs induces neutralising antibodies. Using serum samples from (B), antibody titres capable of neutralising SARS-CoV-2 were determined by microneutralisation (MN) assay. Calculated IC50 doses from multiple serum dilutions are shown. The dotted line shows the LOD.
Effect of liraglutide and RM-493 co-treatment on cholesterol metabolism in DIO mice.Figure 5A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (A) Plasma lipoprotein fractions. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001. A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (B) liver cholesterol levels. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001. A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (C) expression of genes implicated in hepatic cholesterol and lipoprotein uptake. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001. A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (D) expression of genes implicated in cholesterol and bile acid metabolism and excretion were analyzed. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 1(A) TPCK-trypsin is premixed with wtSPINK6 protein or mutSPINK6 protein or PBS in triplicate, incubated with a fluorogenic substrate for 30 min, and then applied to fluorescence assay. Data are presented as mean and SD in a representative experiment performed 3 times. The fluorescence intensity of TPCK-trypsin/PBS mixture is arbitrarily set as 1. ***, P<0.001. (B) At 48h post-transfection of wtSPINK6 plasmid in 293T cells, cell lysate and concentrated supernatant (50×) were applied to the detection of SPINK6 protein and β-actin by WB. (C) At 48h after co-transfection of H7 plasmid with wtSPINK6 plasmid or blank vector, the transfectants were incubated with or without TPCK-trypsin for 30 min, and then applied to examine HA cleavage by WB. Intensities of HA2 and HA0 bands are quantified with ImageJ. HA2/HA0 ratio of each sample is shown at the bottom. (D) At 48h after transfection of H7 plasmid, the transfectants were incubated with TPCK-trypsin in the presence of recombinant wtSPINK6 protein or PBS or a protease inhibitor AEBSF for 30 min and then applied to examine HA cleavage. (E) At 24h after transfection of wtSPINK6 plasmid or blank vector in triplicate, A549 cells were inoculated with H7N9/ah virus. At the indicated hours post infection (hpi), culture media (supernatant) and cell lysate were harvested for viral load detection. Data are presented as mean and SD in a representative experiment performed 3 times. **, P<0.01; ***, P<0.001. Student's t test.
Disruption of hippocampal activity in awake Nrg3-/- mice.Figure 6 A) Examples of local field potential (LFP) recordings (black) from the CA1 region of the hippocampus of a wildtype (wt, left) and a Nrg3-/- mouse (right). Movement of the forepaw was monitored in parallel and is shown on top (blue). Note the appearance of unusual network spikes in the Nrg3-/- mouse (arrows)  B) Example LFP recordings of wildtype and Nrg3-/- mice during paw movement and, below, the corresponding normalized spectrogram of the Fast Fourier Transform (FFT) over time, colored in arbitrary units (A.U.)  C) Population mean FFT from 9 wildtype and 8 Nrg3-/- mice during movement  D) Gamma band activity during paw movement quantified as the area of the FFT from 30-80 Hz  E) Example LFP traces showing sharp wave ripple oscillations from resting wildtype and Nrg3-/- mice  F) Quantification of the occurrence rate of sharp wave ripple oscillations in wildtype and Nrg3-/- mice  G) Population mean FFT of sharp wave ripples in quiet wildtype and Nrg3-/- mice  H) Quantification of peak frequencies of ripples in wildtype and Nrg3-/- mice
H3K122succ can destabilize nucleosomes.Figure 5A Schematic representation of the smFRET assay (top), as well as merged smFRET efficiency histograms of unmodified (middle) and H3K122succ mono-nucleosomes (bottom) measured under indicated NaCl conditions (color coded). The nucleosome population is highlighted by the dashed box. B Fraction of bursts in the nucleosome population (FRET efficiency, E ≥ 0.45) for unmodified (red, open circles) and H3K122succ nucleosomes (turquoise, open circles) plotted against NaCl concentrations and normalized to the respective mean at 0 M NaCl. Mean values (filled circles) were fitted with the Boltzmann sigmoid function (solid lines) to determine the NaCl concentration where half of the nucleosomes have disassembled (x0) as 0.61 M (± 0.04) for unmodified and 0.49 M (± 0.03) for H3K122succ nucleosomes. C Model of H3K122succ function: The establishment of H3K122succ (e.g. by p300/ CBP) favors an open, accessible chromatin state through nucleosome destabilization. Higher DNA accessibility facilitates the binding of the transcriptional machinery and transcription of H3K122succ marked genes. The desuccinylation of H3K122succ, by SIRT5 or SIRT7, results in nucleosome stabilization and reduced transcriptional output.
Figure 2Relative mRNA expression of the Slc2a glucose transporter (GLUT) family members (1-13) in cardiac ECs derived from Vegfb+/+ (n=4) and Vegfb-/- (n=4) mice. Data represent normalized PLIER values (mean ± SEM) from microarray analysis performed on n=4+4 RNA samples (one RNA sample/mouse). mRNA expression levels of the SLC2A transporter (GLUT) family members (1-14) in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments. mRNA expression levels of VEGFB, SLC2A1, NRP1 and FLT1 (VEGFR1) in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments. 2-NBD-glucose uptake in response to 2 h treatment with VEGF-B186 (B186) in HUVECs exhibiting siRNA-mediated knock-down of FLT1, NRP1, SCL2A1, KDR (VEGFR2) or control siRNA. Data presented as mean ± SEM from three individual experiments. Statistical evaluation using one-way ANOVA and Sidak´s multiple comparisons test, p-value: * <0.05 (comparisons made to respective untreated control). Representative images of GLUT1 (left panels) and CD31 (right panels) immunohistochemistry performed on consecutive sections from paraffin embedded mouse hearts. Scale bar, 100 μm. mRNA expression analysis from enriched cardiac endothelial cells (open columns, n=4 mice) or whole heart (closed columns, n= 3 mice). Data presented as mean ± StDev relative to Rpl19 expression. Enrichment factor is calculated as mean cardiac EC/mean whole heart and presented in the box.
Model for the NCOA5‐mediated repression of Abca1 expressionIn response to sterol ligand treatment, LXR–RXR heterodimers initially induce the transcription of Abca1 through recruitment and activation of RNAPII (left).Figure 7Model for the NCOA5‐mediated repression of Abca1 expressionIn response to sterol ligand treatment, LXR-RXR heterodimers initially induce the transcription of Abca1 through recruitment and activation of RNAPII (left). Following prolonged sterol ligand treatment, the NCOA5 repressor directly interacts with LXR at the Abca1 promoter. This repressor complex inhibits the recruitment and function of RNAPII resulting in attenuated expression of Abca1 (right). We hypothesize that prolonged sterol ligand treatment induces a regulatory modification on NCOA5 (shown in green), such as phosphorylation, which facilitates its recruitment to LXR.Crosstalk between pro‐inflammatory TLR3 and anti‐inflammatory LXR pathways promotes the association of NCOA5 with LXR, resulting in the inhibition of RNAPII recruitment and function, and repression of Abca1 gene expression. The regulatory modification of NCOA5 following activation of these two pathways may be similar or distinct to that in (A). We also cannot discount the contribution of additional constituents of this transcriptional complex to the recruitment of NCOA5.
Assessment of immortalisation, tumorigenicity and disseminative potential of the corrected stem cells.Figure 6Upper panel: expression of proteins associated with the acquisition of immortalisation process involved in senescence, in evasion of growth suppression and in apoptosis (Hanahan Weinberg, 2011). The level of P53, P21, RAS and P16 was similar in clone 6, untransduced cells and mass culture from four independent infections, significantly different from the squamous cell carcinoma cell line SCC-13. Lower panel: the phosphorylation state of the PRB restriction point was maintained in clone 6 and untransduced recessive dystrophic epidermolysis bullosa (RDEB) cells, whereas PRB was heavily phosphorylated in transformed cells (SCC-13). Appropriate loading controls were used for each cellular extract (GAPDH for cytoplasmic extracts, histone H3 for nuclear extracts and tubulin for whole-cell extracts. H3 for histone H3, ppRB for hyperphosphorylated pRB and pRB for hypophosphorylated PRB). Transduced clone 6 had a diploid karyotype at passage XVI (see also Fig5A). Transduced clones were not tumorigenic. The tumorigenic potential of the clones was tested by tumour formation in nude mice. Transduced RDEB holoclones (blue) [clone 6 (square, n = 4) passage X, clone 22 (circle, n = 2) passage XI, clone 54 (cross, n = 4) passage X] were not tumorigenic when injected subcutaneously in athymic mice as were untransduced RDEB keratinocytes (passage VII). SCC-13, a squamous cell carcinoma cell line (black) (lozenge, n = 2), was used as a positive control. To test whether the transduced RDEB keratinocytes had disseminated after the generation of an epidermis onto SCID mice (see Fig4C), the internal organs of the recipient mice (3 mice for clone 6 and 2 mice for clone 22) were harvested 385 days post-transplantation and analysed for COL7A1 proviral sequences. No COL7A1 sequence was detected. A mouse transplanted with a holoclone obtained from a healthy donor (YF29) was used as an internal control. PCR-positive controls were genomic DNA from transduced cells (holoclone 6) and cDNAs isolated from healthy keratinocytes. B-actin was run as a loading control.
NCOA5 functions as an LXR corepressorIn vitro pulldown assays using recombinant GST‐LXRα to isolate the indicated in vitro translated NCOA5 constructs.Figure 3In vitro pulldown assays using recombinant GST‐LXRα to isolate the indicated in vitro translated NCOA5 constructs. Assays were performed for 2 h in the presence or absence of 2 μM T0901317. Samples were immunoblotted as indicated. Note the requirement of the NCOA5 NH2‐terminus for interacting with LXRα. Immunoprecipitation of Protein C‐tagged LXRα from stable RAW 264.7macrophagenuclear extracts stimulated with 1 μM T0901317 or vehicle control for 18 h, followed by immunoblotting for endogenous NCOA5 or over‐expressed LXRα. Protein immunoblots of nuclear extracts are shown below. NCOA5 ChIP time course from primary BMMs stimulated with 1 μM T0901317 or vehicle control. qPCR was performed for the Abca1 proximal LXRE. Note the ligand‐stimulated association of NCOA5. Error bars represent ± SEM for n = 4-9 (*P = 0.02). NCOA5 ChIP assays from LXR−/− BMMs stimulated with 1 μM T0901317 or vehicle control for 18 h. qPCR was performed for the Abca1 proximal LXRE. Error bars represent ± SEM for n = 5-6 (**P = 0.0003 for vehicle and **P = 0.002 for T0901317).
Swi6 dimerization and H3K9me2 binding are required for the prevention of H3K9me2 spreading.Figure 5Domain architecture of full‐length Swi6. The indicated mutations in red disrupt the dimerization property of the CSD (L315E) or the ability of the CD to bind H3K9me2 (W104A). Schematic of the IRC1R boundary region. IRC1R partially overlaps with the region that expresses the noncoding RNA borderline (Keller et al, 2013). Positions of the quantitative RT-PCR primers are indicated in red. C-EH3K9me2 levels on heterochromatin‐adjacent genes assessed by ChIP-PCR in swi6+, swi6∆, and swi6‐L315E (CSD dimerization mutant) (C), swi6‐W104A (H3K9me2 binding mutant) (D), and epe1∆ (E) cells. Enrichments over clr4∆ are normalized to adh1+. Average fold enrichment with s.d. is shown for three (C and E) or four (D) independent experiments. P‐values were generated by the Student's t‐test (two‐tailed distribution, two‐sample, unequal variance).