EMBO/sd-character-level-ner huggingface dataset:
NO Impairs Autophagy in AAA Bcl-2 and Jnk1−/− MEFs by Activating mTORC1.Figure 3(A) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated WT Bcl-2 and AAA Bcl-2 MEFs. (B and C) NO donors decreased EGFP-LC3 vesicles in EGFP-LC3-transfected WT Bcl-2 and AAA Bcl-2 MEFs (C). Images were acquired by a confocal microscope (B). WT Bcl-2 and AAA Bcl-2 MEFs were analyzed separately. (D) NO donors increased EGFP-HDQ74 aggregates in EGFP-HDQ74-transfected WT Bcl-2 and AAA Bcl-2 MEFs. WT Bcl-2 and AAA Bcl-2 MEFs were analyzed separately. (E) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated WT Jnk, Jnk1−/−, and Jnk2−/− MEFs. (F) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in HeLa cells. (G) Confocal microscope images of immunofluorescence with anti-phospho-S6 antibody show that DETA NONOate increased S6 phosphorylation; arrowhead shows a cell where this effect was not observed. (H) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in WT Bcl-2 and AAA Bcl-2 MEFs. (I) Confocal microscope images of immunofluorescence with anti-phospho-mTOR and anti-LAMP1 antibodies in HeLa cells show that DETA NONOate increased phospho-mTOR but did not alter its distribution with lysosomes. Graphical data denote mean ± SEM.
Figure 4A-F. Phagocytotic activity of R522 and P522 M-MOP and microglia was assessed using pHrodo Red E. coli and zymosan BioParticles. Phagocytosis arbitrary units (A.U) describes the amount of bioparticle cellular fluorescence emission at each time point. E.coli uptake in M-MOP (A), primary mouse microglia (B), hIPSC-derived microglia (C). Zymosan uptake in M-MOP (D), primary mouse microglia (E) and hIPSC-derived microglia (F). For hIPSC-derived microglia, 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments. All microglia and M-MOP data shows the mean±SD of 3 independent experiments were analysed by two-way ANOVA using Sidak multiple comparison test. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522)
Immunogold Labeling of ERAs with an Antibody Directed against an ER Membrane Marker.Figure 5Immunogold Labeling of ERAs with an Antibody Directed against an ER Membrane Marker(A) Representative section of a cell immunolabeled against a myc-tagged Sec63, an integral ER membrane protein. As a primary antibody, we used a rabbit polyclonal anti-myc and, as a secondary, we used 15-nm gold particles-conjugated anti-rabbit antibody. Nucleus, nuclear envelope, ER, and ERA are indicated as N, NE, ER, and ERA, respectively.(B) High magnification of an electron micrograph of a section of ER. Quantification showed that there are 5 ± 2 gold particles per linear micrometer of ER.(C) High magnification of ERAs. To predict how many gold particles one should expect in a particular ERA, we first calculated and averaged the amount of ER (expressed as length in linear micrometers) present in an ERA (similar to the ones shown in Figure 3B), and normalized the value for its area. These calculations determined that there are 20.8 ± 3.3 μm of ER per μm2 inside the ERAs. These values allowed us to predict how many gold particles would be expected over a section of an ERA if it were packed with ER membranes. Two representative ERAs are shown. The ERA shown in the middle picture should hold 2.4 μm of ER inside and, therefore, should have 12 gold particles. We counted 12 gold particles. The ERA on the right could contain 2.7 μm of ER and should contain14 gold particles; we counted 16 gold particles. The image on the right shows a representative view of a nucleoplasmic region.(D) Quantification of gold-labeling density per area. To assess the signal-to-noise ratio of our immunogold-labeling procedure, we assessed background labeling by counting the number of gold particles over an areas of nucleoplasm (N) and over ERAs, and normalized the counts to the respective areas.
Establishment of FANS for stem cells of the shoot apical meristem (SAM).Figure 1(a) Expression of H2B-mCherry under control of the CLV3 promoter in 14-d-old seedlings. Whole-mount immunostaining using α-mCherry antibodies and laser scanning microscopy (scale bar 10 μm). (b) Example of a FANS experiment: mCherry-positive (+) and mCherry-negative (-) gates of DAPI-gated nuclei. Numbers indicate total number and percent of DAPI (for -) and mCherry (for +) events. (c) A representative example for enrichment of CLV3 transcript in mCherry-positive nuclei determined by qRT-PCR and normalized to wt (N = 1).
Effect of antioxidants in the PD mouse.Figure 7A, B. Co-dosing schedule of rhGAA and NAC (A) and rhGAA and IDE (B). C. GAA activity in tissues from the Gaa KO mouse after treatment with rhGAA alone (black bars) (n of mice = 10) and with co-dosing of rhGAA and 2 gr/Kg/day NAC (n of mice =7, dark grey bars). Data presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each of the tissues. D. GAA activity in tissues from the Gaa KO mouse after treatment with rhGAA alone (black bars) (n of mice =4) and with co-dosing of rhGAA and 100 mg/Kg/day IDE (n of mice =6, light grey bars). Data presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each of the tissues. E E, F. Western Blot analyses of GAA and quantitative analyses of the enzyme in representative tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice =2) or in combination with NAC (n of mice =3) (E) or IDE (n of mice =3) (F). In F Data are presented as mean ± SD. G. Glycogen assay in tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice = 3) or in combination with NAC (n of mice = 3). H. Glycogen assay in tissues from the Gaa KO mouse after treatment with rhGAA alone (n of mice =4) or in combination with IDE (n of mice = 6). Data information: i data are presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each tissue.
Figure 7A global scheme that summarises the role of FXR2P in SE, regulating key molecules of the glutamatergic synapses. KA activates the kainate receptors resulting in ERK1/2 phosphorylation and seizures in both WT and Fxr2 KO mice. While in WT mice, ERK1/2 phosphorylation and protracted seizures maintain, the absence of FXR2P affects glutamatergic signaling, ERK1/2 activity and ultimately the maintenance of seizures.
Figure 1(A) CSAG genes cluster with MAGE-As on chromosome Xq28. (B) Sequence identity between CSAG genes. Note isoforms of CSAG2/3 are splice variants. (C-D) RNA-Seq data from GTEx of the normalized expression of human CSAG1 (C) and CSAG2 (D) in the indicated tissues. Number of biological replicates are as follows: testis 361, brain 255, small intestine 187, white adipose 663, muscle 803, skin 604, esophagus 555, stomach 359, liver 226, heart 429. blood vessel 663, lung 578, colon 373, nerve 619, pituitary 283, blood 755, adrenal gland 258, kidney 85, prostate 245, salivary gland 162, ovary 180, breast 459, pancreas 328, vagina 156, uterus 142, spleen 241, fallopian tube 9, bladder 21, cervix 10. Central band indicates median, boxes define 25 and 75 percentiles, and whiskers define 5 and 95 percentiles. (E-F) Expression of CSAG1 (E) and CSAG2 (F) in normal adjacent (blue) or tumor tissue (red) from TCGA RNA-Seq data. N.D. represents normal adjacent is not available. Data visualized by firebrowse. Number of biological replicates are as follows: ACC 41, BLCA 307, BRCA 524, CESC 175, CHOL 11, COAD 153, COADREAD 232, DLBC 36, ESCA 121, HNSC 416, KICH 46, KIPAN 515, KIRC 411, KIRP 58, LAML 7, LGG 528, LUAD 301, LUSC 400, MESO 31, OV 183, PCPG 70, PRAD 204, READ 79, SARC 156, SKCM 422, STAD 288, STES 409, TGCT 126, THCA 408, THYM 75, UCEC 306, and UCS 37. Central band indicates median, boxes define 25 and 75 percentiles, and whiskers define 5 and 95 percentiles. (G) Xq28 MAGE and CSAG genes are frequently co-expressed in lung squamous carcinoma tumors (n=184). RNA-Seq gene expression data analyzed from TCGA. Statistics shown were calculated by linear regression analysis. (H-K) Expression of CSAG1 (H-I) or CSAG2 (J-K) in patients from indicated cancer types correlates with poor overall survival. Data is from TCGA and visualized by kmplotter.
Figure 3Left panel, (M/E)2 dimer (PDB 5O6A, [10]) colored as in Figure 1. For clarity, residues downstream E Gln404 were deleted, and only residues 1 to 20 of M are shown in tube representation. The framed region is enlarged in the middle panel, with the pre-fusion E/M heterodimer (E: grey; M: brown) and the post-fusion E trimer (color-coded as domains) superimposed on helices αA and αB, illustrating the common site occupied by M and by the E stem at different stages of the flavivirus cycle. E and M run in opposite directions within the αA/B groove, interacting with the same domain II residues. Right panel: close-ups of the interactions with residues displayed as sticks. Top, residue Ile4 of M takes the place of Phe403 of the stem. The bottom panel illustrates the alternative interactions made by the strictly conserved Trp219 side chain with the main chain of M in pre-fusion and of the stem in post-fusion forms. In both, top and bottom panels, stem and M residues are labeled in green and brown, respectively. pH-dependent dissociation of E and M. TBEV preparations treated at pH 8 (left) and pH 6 (right) were solubilized with Triton X-100 and subjected to sucrose gradient centrifugation. The fractions were analyzed by SDS-PAGE and densitometry. Representative examples of at least two independent experiments are shown. Positions of E dimers (D) and E trimers (T) in the gradients are indicated.
Figure 6(A,B) AMG386 (5.6mg/kg s.c.; twice/week) and SHP099 (a: 200 mg/kg; b: 100mg/kg orally/daily) were administered individually or together to B16F10 tumor-bearing mice (A: control n=7; SHP099: n=8; AMG386: n=8; SHP099+AMG386 n=11; B: control, SHP099 and AMG386: n=4; SHP099+AMG386: n=8). Panels depict tumor growth from initiation of treatment to endpoint (left) and tumor weight (right) at endpoint; error bars ± s.d.; P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001. (C,D) p-TIE2 in tumor vessels from control and AMG386-treated mice; representative confocal images; arrowheads point to CD31+/p-TIE2- endothelial cells (scale bars: 50μm) (C), and quantification (D) of p-TIE-2/CD31+ in control (n=3) and AMG386 treated (n=3) tumors (1,200 CD31+ cells/group). P values from two-tailed Student's t-test; ***P<0.001. (E,F) p-EphrinB (E) in representative tumor vessels from control (n=3) and AMG386-treated (n=3) tumors; confocal images (E), (scale bar: 50μm) and quantification of p-EphrinB/CD31+ (F); P values from two-tailed Student's t-test; ***P<0.001. (G) Vessel perfusion visualized by FITC-dextran (green); CD31 (red) identifies the endothelium; DAPI (blue) identifies the nuclei; representative tumor confocal images (scale bar:50μm) of 3 mice/group; arrow heads point to FITC-dextran extravasation; asterisks to extravasated erythrocytes. (H) Quantification of cleaved caspase-3 in the tumor vasculature (CD31+ cells); n=3 tumors/group; number of CD31+ cells counted: control (n=311); SHP099 (n=425); AMG386 (n=451); combined SHP099+AMG386 (n=484). Error bars: ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001. (I) Quantification of tumor vascular area; n=5/tumors group. Error bars: ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001. (J) Quantification of "vascular sleeves" (CD31-Collagen IV+) n=5/tumors group. Error bars: ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001. (K, L) (K) % necrotic tumor area (n=4/tumors group) and (L) % proliferating tumor cells (n=3/tumors group) in control and treated groups. Error bars: ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; *P<0.05, **P<0.01, ***P<0.001. (M) Representative control and treated (n=5/group) B16F10 tumor sections through the maximum diameter; H&E staining; (scale bars: 10mm). (N) Proliferating Ki67+ cells (green) in representative control and treated tumors (n=3/group); CD31 identifies the vessels; DAPI identifies cell nuclei. Confocal images (scale bar: 50μm).
Figure 9Two modes of collaboration between N- and C-degron pathways in protein removal. Competition between Gly2 myristoylation and Gly/N-degron pathway-mediated degradation.
Figure 3(a) Any cortical surface of living organisms could be considered a structure under tension embedded in viscous fluids, where the stresses in the three dimensions at each point are locally balanced (b) Representation of an Stokeslet pair, at the limits of a perpendicular section of a planar hydrodynamic active cortex (red shadow), pointing in opposite directions (white arrows) and their associated flow lines (black) and mechanical pressure fields (color-coded). (c) Divergence fields of Stokeslet dipoles of opposite magnitudes corresponding to cortical expansion or contraction (left). These dipoles carry an associated mechanical pressure (middle) and power (right). Mechanical power is positive at the central Stokeslet pair that is surrounded by a cortical ring that resists its deformation. (d) Pipeline describing the different steps involved in HR. From time-lapse 2D videos (green shadowed in yellow) a set of parameters for the fluid phase (blue) are sequentially acquired through PIV and regression analysis, in particular the fluid stress (shadowed in pink). From these, the relative elastic stress and mechanical power (purple shadowed in pale blue) of the cortex are extracted for different geometrical configurations (e) Representation of the planar cortex (PC) (left) and the spherical cortex (SC) (right) models where the elementary solutions (dots) pointing in opposite directions (pale green arrows) are evenly distributed on a square grid (PC) or symmetrically distributed around the main cylindrical axis of a sphere (Nc = 16) (SC). For the PC model only the external components are shown in one row and one column. For the SC model only the external components are shown in one column.
Figure 3Representative cortical images of MXO4 staining for APP and APP/Nlrp3-/- 15 months old mice. Scale bar: 50µm. Cortical amyloid plaque number and size quantification, and Amyloid-beta1-40 and 1-42 ELISA quantification for APP and APP/Nlrp3-/- mice (5 and 15 month old)
a) Diagram of AR truncation constructs.figf6(a) Diagram of AR truncation constructs. N-term, N-terminal; C-term, C-terminal; m1-m4, mutants 1-4; NTD, N-terminal domain; DBD, DNA binding domain; Hinge, hinge domain; LBD, ligand binding domain. (b) HEK293 cells were transfected with TFEB and influenza hemagglutinin (HA)-tagged full-length AR25 (FL) or a C-terminal AR mutant (m1, m2, m3, m4). Immunoprecipitation (IP) was performed with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown). (c) HEK293 cells were transfected with TFEB and HA-tagged N-terminal AR mutant containing a 19Q or 112Q tract (NT19Q, NT112Q). Immunoprecipitation was performed as in b. with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown). (d) MN-1 WT, MN-1 AR24Q and MN-1 AR65Q cells were cultured in standard medium (Ctl) or in medium supplemented with R1881 and then immunostained with antibodies to AR and to TFEB. Cells were scored for nuclear or cytosolic localization of TFEB. Scale bars, 20 μm. (e) Quantification of d. Untreated, F = 0.56; R1881, F = 5.51; ANOVA with post hoc Tukey test. **P 0.01. (f) We transfected MN-1 WT cells with the 4X-CLEAR luciferase reporter and either BFP-empty or AR25Q-BFP, under standard culture conditions (control), ammonium chloride treatment or rapamycin treatment. Control, t(3) = 15.73; NH4Cl, t(4) = 4.108; rapamycin. t(4) = 4.58; t-test. **P 0.01. (g) We transfected MN-1 AR24Q cells with either scrambled shRNA control vector or AR shRNA vector and measured 4X-CLEAR luciferase reporter activity under baseline conditions (control) or after rapamycin. Control, t(4) = 0.51; rapamycin, t(4) = 7.15; t-test. **P 0.01. (h) RT-PCR analysis of MN-1 AR24Q cells transfected with scrambled shRNA control or AR shRNA vector: Gla, t(4) = 4.77; Atp6v1h, t(4) = 3.75; Mcoln1, t(4) = 13.74; Ctsd (cathepsin D), t(4) = 7.91; and Lamp1, t(4) = 6.46; n = 3 independent experiments, *P 0.05, **P 0.01, t-test. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.
Figure 3A-F Double immunolabeling of PDS5B (green) and SYCP3 (red) in a metaphase I spermatocyte (A-B) and selected bivalents (C-F). The sex bivalent (XY) is indicated. G-J Double immunolabeling of PDS5B (green) and kinetochores (ACA, red), and staining of the chromatin (DAPI) in a metaphase II spermatocyte.
Figure 5A and B. Representative transmission EM pictures of RBEWT/WT (A) and RBEKO/KO (B) photoreceptor ribbon synapses (blue rectangles, area adjacent to synaptic junction used for measuring junction-associated vesicle density [size = 200 nm x 300 nm]; black squares, are in the presynaptic cytosol used to measure cytosolic synaptic vesicles [size = 200 nm x 200 nm]).C. Average vesicle ratio (expressed as vesicle numbers in the area adjacent to synaptic junctions (blue rectangle) or the cytosol (black square) normalized to RBEWT/WT.D. Average number of docked vesicles within a 150 nm range in the vicinity of the active zone.
Figure 3(A) Comparative permissiveness of various organs from B6.K18-hACE2IP-THV and B6.K18-ACE22prlmn/Jax transgenic mice (n = 5-6/group) to SARS-CoV-2 replication, as determined at 3 dpi by conventional E-specific or sub-genomic Esg-specific qRT-PCR. Red lines indicate the qRT-PCR limits of detection. (B) Comparative quantitation of hACE-2 mRNA in the lungs and brain of B6.K18-hACE2IP-THV and B6.K18-ACE22prlmn/Jax transgenic mice (n = 5-6/group). Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p <0.01, ns = not significant). (C) Heatmaps represent log2 fold change in cytokine and chemokine mRNA expression in the lungs or brain of B6.K18-hACE2IP-THV and B6.K18-ACE22prlmn/Jax mice at 3 dpi (n = 5-6/group). Data were normalized versus untreated controls.
Figure 4Schematic of the dsDNA curtain assay used for homology search assays. Schematic of the Atto565-labeled tailed duplex substrate and PSC assembly procedure for homology search assays. Widefield images of Atto565-labeled PSCs (magenta) bound to dsDNA curtains stained with YOYO1 (green). The PSCs were prepared with the indicated combinations of Rad51, Rdh54 and GFP-Rad54.   Number of Atto565-labeled PSC binding events per dsDNA molecule for PCS prepared with Rad54 and Rdh54 only (minus Rad51 ; N = 372), Rad51 plus Rad54 only (N= 117), Rad51 plus Rdh54 only (N = 258), Rad51 plus Rdh54 and Rad54 (N = 303). The error bars represent the s.d. for three independent experiments.
Characterization of skeletal muscle of SGK1 transgenic mice.Figure 5A. H&amp;amp;E staining of tibialis anterior sections from 2‐month old control and transgenic mice shows no differences (scale bar 90 µm). B. Morphometric analyses of minimum Feret's diameter of TA muscle reveal no changes in fiber size distribution or mean fiber size. C. Western blots and densitometric analysis of muscle from transgenic and control mice. Downstream targets of the mTOR signalling cascade (p70S6K and 4EBP1) demonstrate significant upregulation of their phosphorylated forms in sgk1tg mice. D. Western blot analyses and densitometry of phosphorylated FOXO3a and total FOXO3a of tibialis anterior muscles from control and transgenic mice (n = 4) show an increase of phosphorylation at S315 and T32.
Figure 2A. TRAPPII and TRAPPIII complexes were immunoprecipitated by antibodies specifically recognizing TRAPPC9 or TRAPPC12, and the presence of various co-precipitating TRAPP subunits was detected by immunoblotting. B-D. TRAPP stimulated guanine nucleotide exchange on purified Rab18, Rab1 and Rab2 GST fusion proteins measured by release of 3H-GDP that was pre-loaded onto the Rab proteins. E-G. Nucleotide exchange reactions measured by time-dependent binding of [35S]GTPγS. Immunoprecipitates using the indicated antibodies were included in the assay as the GEF to be tested. In the 3H-GDP release experiments, the reactions were incubated for 30 minutes at 37°C before the Rab proteins were subjected to nitrocellulose filter binding. In the [35S]GTPγS binding experiments, the reactions were incubated for the indicated amount of time before filter binding. H-I. Anti-Myc antibody immunoprecipitates from lysates of HEK293T cells transfected Myc-TRAPPC9 were used as the GEF for GST-Rab1 or GST-Rab18 in 30 min incubations at 37°C with [35S]GTPγS.
HCV infection stimulates ATF4, Parkin, and PINK1 gene expression.Figure 3(A and B) Quantitative analyses of ATF4, Parkin, and PINK1 gene expression in HCV-infected cells. Huh7 cells infected with HCVcc for 5 days or treated with CCCP (10 or 20 µM) for 24 h were used for analysis of ATF4, Parkin, and PINK1 gene expression. (A) Intracellular mRNA levels of ATF4, Parkin, and PINK1 were analyzed by real-time qRT-PCR. GAPDH was used to normalize changes in Parkin and PINK1 gene expression (mean ± SD; n = 3; *p<0.01, **p<0.05). P values were calculated by using an unpaired Student's t-test. B) The protein expression levels of Parkin and PINK1 were analyzed by immunoblotting with anti-Parkin and PINK1 antibodies. The expression of HCV core protein was analyzed by immunoblotting with anti-HCV core antibody. β-actin was used as an internal loading control. (C) Western blot analysis of Parkin expression in liver biopsies of chronic hepatitis C patients. Samples 1 and 2 represent liver biopsies of HCV-negative patients and samples 3-7 represent HCV-positive chronic hepatitis patients. (B and C) The relative intensity of Parkin and PINK1 expression normalized to β-actin analyzed by ImageJ. (D) Confocal microscopy showing Parkin overexpression in HCV-infected cells. Huh7 cells infected with HCVcc were immunostained with anti-Parkin antibody (red). Fluorescence image of HCV E2 verifies HCV-infected cells (light gray). Nuclei were stained with DAPI (blue). Infected (+) and uninfected cells (−) are marked.