EMBO/sd-character-level-ner huggingface dataset:
Figure 5Outline of the RAP-MS experiment. Enrichment of the AcGFP mRNA using the antisense biotinylated probes. The NORAD lncRNA is used as a control abundant RNA. Western blot for the protein indicated below the blot, comparing input MCF-7 cells, and IP with IgG or the indicated antibody. The arrows indicate the expected size of the protein of interest. RIP-qPCR for the indicated RNA (AsGFP or NORAD) in cells transfected with the AcGFP bearing NucLibB in the 3' UTR, with RIP performed with the indicated antibody and RNA levels determined by qRT-PCR. N=5. Error bars - s.e.m. Correspondence between binding of the indicated pairs of proteins to NucLibB tiles. Spearman's correlation coefficient is indicated. Color indicates point density. Same as (E), for NucLibC tiles that contained sequence variants of the Jpx#9 tile. Comparison of IP/Input ratios for the indicated factors, for NucLibB tiles containing the SIRLOIN element (with up to 6 mismatches) and other tiles. Same as 2B, for IP/Input ratios for the indicated proteins. Correspondence between binding of the indicated protein and the Nuc/Cyto ratios induced by NucLibB tiles. Nuc/Cyto ratios for NucLibB tiles, bound (+, log2(IP/Input)≥0.5) or not bound (-, log2(IP/Input)<0.5) by the indicated factors. Boxplots are as in Figure 1C. P values computed using two-sized Wilcoxon rank-sum test. IP/Input ratios for the indicated factors, for ten Jpx#9 sequences with shuffled sequences in bases 80-109, WT sequence of Jpx#9 or 3 repeats of the SIRLOIN element ('3X SIRLOIN'). Boxplots are as in Figure 1C.
Figure 8(A-F) Forebrain slices were pre-incubated for 30 min with vehicle (water) or 10 μM Myr-Pep2 or Myr-DSPL. Like Myr-DSPL (Joiner et al., 2010), Pep2 was myristoylated at its N terminus (Myr-Pep2) to make it membrane permeant. Slices were then treated with ISO (10 M, 5 min) or vehicle (water) before solubilization, ultracentrifugation, and IP of β2AR (A-C) or simultaneously α11.2 and GluA1 with a combination of corresponding antibodies within same samples (D-F).(A) Myr-Pep 2 displaced α11.2 (lane 5 vs. 3, top of blot) but not GluA1 (middle, same blot) from β2AR (bottom, same blot); the inverse was true for Myr-DSPL (lane 1 vs. 3).(B,C) For quantification, α11.2 and GluA1 immunosignals were normalized to β2AR signals (**p<0.01, ***p<0.001, One Way ANOVA). (A-F) Forebrain slices were pre-incubated for 30 min with vehicle (water) or 10 μM Myr-Pep2 or Myr-DSPL. Like Myr-DSPL (Joiner et al., 2010), Pep2 was myristoylated at its N terminus (Myr-Pep2) to make it membrane permeant. Slices were then treated with ISO (10 M, 5 min) or vehicle (water) before solubilization, ultracentrifugation, and IP of β2AR (A-C) or simultaneously α11.2 and GluA1 with a combination of corresponding antibodies within same samples (D-F).(D) Myr-Pep2 blunted ISO-induced phosphorylation of α11.2 S1700 (lane 6 vs. 4, top of blot) but not GluA1 S845 (middle, same blot); the inverse was true for Myr-DSPL (lane 2 vs. 4).(E,F) For quantification, pS1700 and pS845 immunosignals were normalized to α11.2 and GluA1 signals, respectively (***p<0.001, One Way ANOVA). (G) Representative cell-attached recordings from hippocampal neurons as in Figure 7. In interleafed experiments cultures were pre-incubated for 30 min with 10 M Myr-Pep2 or scrambled Myr-Pep2 (Myr-Pep2scr). The patch pipette contained either vehicle (H2O; control) or 1 µM ISO. The ISO induced increase in NPo was prevented by Myr-Pep2 but not Myr-Pep2scr. Arrows indicate the 0-current level (i.e. closed channel).(H) Summary plot for (G). For statistical analysis, the NPo value was determined for each recording and pooled under each condition for comparison (n=10-12 patches; *p<0.05, One Way ANOVA with Tukey post-hoc test).
Prazosin inhibits glioblastoma growth in immunocompetent mice.Figure 3A. Viability analysis of GL261 treated with prazosin in vitro. *P=0.0286, n=4, two-sided Mann-Whitney U-test. B. Schematic representation of prazosin treatment in vivo of glioblastoma-bearing immunocompetent mice. C. Prazosin inhibits in vivo tumorgrowth in an immunocompetent model of glioblastoma. Left panel: Bioluminescent in vivo images of tumors in C57/Bl6mice treated with vehicle or prazosin (PRZ) for 14 days.Middle panel: Quantification of the bioluminescent signals. *P=0.0002, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice treated with prazosin (PRZ) or vehicle. log-rank Mantel-Cox test. The treatment period is shaded in gray. D. Hematoxilin/eosin staining of C57/Bl6 mice brain coronal sections sacrificed after 14 days of treatment with prazosin. Scale bar = 2 mm. E. Fluorescence imaging of GL261 intra-striatal grafts at 1 and 2 h after an intra-peritoneal injection of BODIPY FL prazosin, the green-fluorescent derivative of prazosin. Scale bar = 2 mm.
Figure 2(A) Representative phase contrast, yellow fluorescent (indicating p53-Venus levels), and red fluorescent (indicating reporter mCherry levels) images at the indicated time points for MDM2 promoter reporter cells exposed to the "natural dynamics" Nutlin-3 dosing regimen. (B-G) Single cell traces of mCherry expression for the "responding" (light gray) or "not responding" (dark gray) MDM2 promoter or CDKN1A promoter cells exposed to the natural dynamics (B), low amplitude (D), high amplitude (D), high frequency (E), low frequency/short duration (F), or long duration (G) Nutlin-3 dosing regimens. The average trace for responding and not responding cells is shown in red and blue, respectively. Heat maps show alternative representation of all single cell traces below each associated time course plot.
Figure 1A, B Bottom panels: gene annotation track (mm10); the activated gene is marked in green and the location of the TALE-VP64 target sequence is shown by the vertical dashed green line. Middle panels: DamID tracks of NL interactions in control cells ("control", blue line) and cells expressing TALE-VP64 ("experimental", red line). n indicates the number of independent biological replicates that were combined. Noise was suppressed by a running mean filter of indicated window size. Shading between the lines corresponds to the color of the sample with the highest value. Arrowheads on the right-hand side mark the 5th and 95th percentiles of genome-wide DamID values. Top panels: domainograms; for every window of indicated size (vertical axis) and centered on a genomic position (horizontal axis) the pixel shade indicates the ranking of the change in DamID score (experimental minus control) in this window compared to the genome-wide changes in DamID scores across all possible windows of the same size. Blue: DamID score is highest in control samples; red: DamID score is highest in experimental samples (color key on the right of panel (a)). In (a) activation of Sox6 was the experimental perturbation, activation of Nrp1 (which is located on a different chromosome) served as control; in (b) activation of Nrp1 was the experimental condition and activation of Sox6 served as control.
Figure 6Upon being complexed with sgRNA, SaCas9 confers stable binding to the DNA target when 6 PAM-proximal matches exist. It then triggers sequential DNA unwinding from the PAM-proximal region to the PAM-distal end and samples adjacent to the DNA for guide RNA complementarity. More than 18 bp RNA-DNA matches close to the PAM allow complete R-loop formation and endonucleolytic cleavage of both DNA strands. Afterward, SaCas9 remains bound to the PAM while autonomously releasing the PAM-distal DNA. The interplay between SaCas9 and the DNA is mediated by the post- and pre-PAM interactions, as indicated by the blue and black triangles, respectively.
Figure 3A. U1 cells were treated with 50 ng/μL of Vs for 15 min (grey area), washed, lysed and kinetics of Vs internalization and retention (yellow area) was quantified by subjecting cell lysate and supernatant to ICP-MS for measuring intracellular and extracellular Vs, respectively. B. Schematic representation showing working principle of Orp1-roGFP2 biosensor. C. U1-Orp1-roGFP2 cells were treated with Vs for 15 min, followed by exposure to H2O2 for 2 min, and ratiometric response was measured. D. Schematic representation showing working principle of Grx1-roGFP2 biosensor. E. U1-Grx1-roGFP2 cells were pre-treated with Vs for 15 min, exposed to H2O2 for 2 min, and ratiometric response was measured. F. U1-Grx1-roGFP2 cells were treated with two doses of 50 μM H2O2 (black arrows) and the ratiometric response was measured (blue line). Parallelly, U1 cells treated similarly with H2O2 were exposed to Vs at the indicated time point and the ratiometric response was measured (green line). **** P<0.0001, by Wilcoxon matched-pairs signed rank test. G, H. U1 Grx1-roGFP2 cells were supplemented with BSO (G) or GSH (H) for 16 h to deplete or replenish GSH, respectively. Following this, cells were treated with Vs for 15 min, exposed to H2O2, and the ratiometric response was measured.
Figure 1A. Representative pseudocolor images of PC6 cells transfected with EYFP-Parkin (green) and mitochondrially targeted CFP (red) with and without PINK1 and Miro1 shRNA and Miro2 shRNA. Zoomed images show translocation of EYFP-Parkin to mitochondria in PINK1-overexpressing PC6 cells B. Quantification of EYFP-Parkin translocation to mitochondria. The percentage of PC6 cells with EYFP-Parkin translocated to mitochondria in response to PINK1 overexpression is lower in the Miro shRNAs-expressing groups when compared with the scrambled shRNA-expressing group (scr shRNA). ****P < 0.0001, n = 6 dishes per group, 20 fields per dish, one-way ANOVA C. The kinetics of EYFP-Parkin translocation is different in PC6 cells transfected with scrambled or Miro shRNAs. Cells were plated in separate compartments of the same dish, enabling us to visualize Parkin translocation simultaneously over 3 h under similar conditions in response to antimycin with oligomycin treatment (A&O, both 10 µM). The spatial heterogeneity of EYFP-Parkin (coefficient of variation of the intensity of individual pixels) was estimated for individual cells for each time point (n = 39-47 cells; P < 0.0001 difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM) D. Miro shRNAs mitigate the effect of A&O on EYFP-Parkin translocation. PC6 cells transfected with EYFP-Parkin with and without Miro shRNAs were treated with DMSO or A&O (both 10 µM) for 3h. The figure demonstrates that the percentage of cells with Parkin translocated to mitochondria was lower in the Miro shRNAs-expressing group when compared with the scrambled shRNA-expressing group. ****P < 0.0001, n = 4 dishes, 20 fields per dish, one-way ANOVA E. Miro shRNAs inhibit PINK1-induced mitophagy in PC6 cells. The figure shows the changes in the excitation spectra of mitochondrially targeted pH-sensitive Keima (ratio of 561 nm/458nm fluorescence intensities). ****P < 0.0001, n = 80 cells from 4 dishes, Kruskal-Wallis test F. Miro shRNAs inhibit basal mitophagy in primary cortical neurons expressing mitochondrially targeted Keima. Mitochondria in lysosomes (acidic pH) were counted manually, by a blinded observer (**P < 0.01, n = 9 dishes, 9 fields per dish, from 2 independent experiments, one-way ANOVA) G. Miro shRNAs decrease the number of mitochondria co-localised with the autophagosome marker EGFP-LC3B in primary cortical neurons (**P < 0.01, n = 5 dishes, 9 fields per dish, one-way ANOVA).
Dual anti-Ang-2 and anti-VEGF therapy acts synergistic on vascular normalization and macrophage infiltration in experimental GBM.Figure 4Figure 4: Dual anti-Ang-2 and anti-VEGF therapy acts synergistic on vascular normalization and macrophage infiltration in experimental GBM. Immunofluorescence staining of CD31 and desmin in GL261 glioblastoma after single and dual treatment with anti-Ang-2 (AMG386) and anti-VEGF (aflibercept) (A). Corresponding quantitative analysis of microvessel densities and pericytes numbers (B; control n=24; AMG386 n=10; aflibercept n=12; AMG386 + aflibercept n=13). . Staining for mouse IgG (red) as a surrogate parameter for vascular leakage and CD31 (green) is shown in GL261 glioblastoma sections following different anti-angiogenic treatments (C) with quantification in (D; n=3). Quantification of necrotic areas in GL261 glioblastoma sections after anti-angiogenic treatment is shown as percent of whole tumor area (E; control, aflibercept, AMG386 + aflibercept n=4; AMG386 n=3). Analysis of Glut1 immunoreactivity in GL261 glioblastoma sections (F; n=3). Double-immunofluorescence stainings of macrophages (F4/80) and tumor vessels (vWF) in mouse GBM after treatment with AMG386, aflibercept or the combination of both are shown in (G) Quantitatve analysis of tumor infiltrating leukocytes (F4/80+, CD3+, Ly-6G+) following anti-angiogenic treatment is displayed in (H; control n=28; AMG386 n=12; aflibercept n=11; AMG386 + aflibercept n=12). Kaplan-Meier survival analysis of GL261 tumors grown in C57BL/6 mice following anti-angiogenic treatment (I; control n=38; AMG386 n=13; aflibercept n=12; AMG386 + aflibercept n=13). Double-immunofluorescence stainings with anti-F4/80 and anti-CD206 in brain tumor sections of mice treated with anti-Ang-2 (AMG386), anti-VEGF (aflibercept) or the combination of both are shown in (J). Corresponding quantitative analyses of tumor infiltrating F4/80+ cells and CD206+ cells, and the ratio of CD206+ vs. F4/80+ upon anti-angiogenic therapy are displayed in (K; control n=21; AMG386 n=13; aflibercept n=9; AMG386 + aflibercept n=4). Flow cytometry of tumor infiltrating macrophages following enzymatic dissociation of mouse GL261 brain tumors plus/minus anti-angiogenic therapy. Percent of CD206+ (L) and MHC class IIHI cells (M) among CD45+CD11b+GR1-F4/80+ cells are displayed (L,M; control n=4; AMG386 n=4; aflibercept n=5; AMG386 + aflibercept n=5). Statistical analyses were performed using one-way ANOVA and Tukey's multiple comparison except for Log-rank and Wilcoxon (I) and Kruskall Wallis (B). * p < 0.05, ** p < 0.01, *** p < 0.005, # p < 0.005 of aflibercept + AMG386 vs aflibercept; data are mean ± SEM (A-K)
a) Mitochondria from MEFs from Parl WT (+/+) and KO (−/−) mice were treated with the indicated doses of proteinase K (Prot.f2(a) Mitochondria from MEFs from Parl WT (+/+) and KO (−/−) mice were treated with the indicated doses of proteinase K (Prot.K) for 30 min on ice and then immunoblotted for the indicated proteins. Full-length and cleaved PGAM5 bands are indicated with arrows. (b) Pgam5 WT and KO MEFs were transduced with either nonspecific (NS) or Parl shRNA and either treated (+) or not (−) with CCCP for 3 h. Mitochondria were then purified and analysed by immunoblot for the indicated proteins. (c) WT PINK1 as well as kinase dead (KD) or Parkinson's disease-associated PINK1 mutants (C92F, A168P, H271Q, G309D, L347P, G386S, R464H) were co-transfected into HEK293T cells with the same amount PGAM5 (lane 1-9) or GFP (lane 10-18) expression plasmids and total cell lysates were immunoblotting for PINK1, PGAM5 and actin. The ratio of WT full-length (63 kDa) versus cleaved (54 kDa) PINK1 (*/#) is shown. (d) Anti-myc immunoprecipitates (IP) of lysates from 293T cells transfected with combinations of myc-tagged PGAM5 and PINK1-V5 with or without CCCP treatment and immunoblotting with anti-PINK1 or anti-myc. (e) Four constructs expressing either: (1) no PGAM5 (empty vector) or (2) amino acids 1-98, (3) 1-110 and (4) 98-289 truncated versions of PGAM5 (left panel). Immunoblot of HEK293T cells expressing PINK1-GFP and myc-tagged PGAM5 truncation mutants as indicated with anti-PINK1 or anti-myc for the input proteins as well as the anti-GFP IP (right panel). (f) Co-transfection of GFP, WT PGAM5, 2RA mutant (R98A and R104A) or the 2HA mutant (H99A and H105A) with a PINK1 expression plasmid into HEK293T cells and the input cell lysates as well as the anti-myc IP were immunoblotted with antibodies against PINK1, PGAM5 or actin. WT PINK1 was expressed in HEK293T cells with or without PGAM5 or a GFP control plasmid after (g) DMSO or (h) CCCP treatment. Cells were permeabilized with digitonin or Triton X-100 separately, followed by treatment with the indicated amounts of trypsin on ice for 30 min. Cell lysates were immunoblotted for PINK1, PGAM5, Tomm20 or HSP60 as indicated. Full-length (*63 kDa) and cleaved (#54 kDa) PINK1 are shown.
Figure 6A Theoretical calculation of the rescaled radius of paraspeckles versus the rescaled transcription rate for the case in which the A and C blocks were deleted by CRISPR/Cas9 in the steady state. The graph is derived by assuming that NEAT1_2 is produced at a constant rate. The produced NEAT1_2 RNPs diffuse in the solution and the free diffusion is hindered by the attractive interactions between NEAT1_2 RNPs with the interaction parameter χ (we used χ = 1.0). NEAT1_2 was degraded at the constant rate k0. The radius was rescaled by the length scale $\sqrt{D/k_{0}}$ and the transcription rate was rescaled by the inverse time scale k0(D/k0)3/2/v0. B Quantitation of NEAT1_2 by RT-qPCR in WT and ∆5′/∆3′/∆PAS cells with or without MG132 treatment (6 h). Data are represented as mean ± SD (n = 3). C The paraspeckles in WT and ∆5′/∆3′/∆PAS cells detected by SRM using NEAT1_5′ and 2k FISH probes (green), and NONO IF (magenta) under the conditions shown in (B). Scale bar, 500 nm. D Quantitation of the diameter (Sx) observed by SRM in WT and ∆5′/∆3′/∆PAS cells under the conditions shown in (B). WT [mean size: MG132 (-), 354.2 nm (n = 32); MG132 0.25 μM, 380.5 nm (n = 68); MG132 1 μM, 394.1 nm (n = 108); MG132 5 μM, 409.6 nm (n = 103)], ∆5′/∆3′/∆PAS [mean size: MG132 (-), 344.4 nm (n = 18); MG132 0.25 μM, 472.1 nm (n = 27); MG132 1 μM, 601.4 nm (n = 27); MG132 5 μM, 721.2 nm (n = 26)]. (*: P = 0.029, ****: P < 0.0001, compared with WT: Kruskal-Wallis test with Dunn's multiple comparison test). Each box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top). E Theoretical calculation of the fraction of A (or C) blocks in the shell vs the logarithm of the transcription rate (rescaled by the rate at which the transcripts are spontaneously incorporated in the paraspeckle). The numbers of segments of A blocks were 5.0 (cyan), 7.5 (orange), 10.0 (green), and 12.5228 (black). The parameters used for the calculations are shown in the Materials and Methods. F Quantitation of NEAT1_2 by RT-qPCR in ∆0-0.8 kb cells with or without MG132 treatment (5 μM for 6 h). Data are represented as mean ± SD (n = 3). G The paraspeckles detected by SRM with NEAT1_5′, 1k, and 2k (green), and NEAT1_3′ (magenta) FISH probes in the ∆0-0.8 kb cells under the conditions shown in (H). Scale bar, 500 nm. H Graph showing proportion of paraspeckles with localization of the NEAT1 5′ ends to the core and shell or the shell in ∆0-0.8 kb cells under the conditions shown in (F) [MG132 (-): n = 78, MG132 (+): n = 127].
Genetic interaction between kinesin‐mediated intracellular trafficking and cellular proteolytic pathways mediating necrotic cell death.Figure 6Genetic interaction between kinesin‐mediated intracellular trafficking and cellular proteolytic pathways mediating necrotic cell death. Dysfunction of either kinesin 1 heavy chain (UNC‐116) or the monomeric kinesin UNC‐104 does not significantly enhance suppression of neurodegeneration in aspartyl protease‐deficient mutant animals (A, B), in animals with compromised lysosomal acidification (C, D), or in calpain protease‐deficient mutants animals (E, F). Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t‐test).
Modelling the three‐dimensional structure of Atg8.Figure 1(A) Multiple sequence alignment of Atg8 homologues. Amino‐acid sequences of Atg8 and its mammalian homologues are aligned using the Clustal_W program. Underlined residues represent mutated amino acids in Atg8. (B) Alignment of Atg8 predicted three‐dimensional structure with its mammalian homologues. SWISS MODEL was used to predict Atg8 structure and PyMol was used for presentation. Orange represents the predicted Atg8 structure, and red, blue and green represent GATE‐16, GABARAP and LC3, respectively. (C) Three‐dimensional model of Atg8. The predicted structure contains a ubiquitin fold decorated by two further amino‐terminal helices (amino acids Tyr 49, Leu 50, Phe 77 and Phe 79 are marked in green).
Figure 1A, schematic of residual MDR cell generation and subsequent analyses. The circular arrow represents the DTP state. B, phase contrast images of the morphological changes in MDR cells, residual MDR cells and regrown cells for Du145TXR or MCF-7ADR. Red circles mark magnified areas (bottom). Scale bars, 50 μm (low magnification images). C-D, cell viability of residual MDR cells (RMCs) and MDR cells (MCs) of (C) Du145TXR or (D) MCF-7ADR cells using the same combination treatment for 24 hours on the indicated days. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments. E, cell viability of regrown cells (RCs) and Du145TXR (left) or MCF-7ADR (right) cells with the same combination treatment for 24 hours. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments. F-G, drug tolerant rate of (F) Du145/Du145TXR (20 nM taxol/20 nM taxol plus 50 μM verapamil) or (G) MCF-7/MCF-7ADR (200 nM adriamycin/200 nM adriamycin plus 50 μM verapamil) cells treated with drugs on the indicated days. Drug tolerant rate defined by comparing cell viability followed treatment, RMCs VS. MCs or CCs VS. PeCs, > 1 representing drug tolerant. Induction Time indicates the time of entering DTP state; Sustaining Time indicates the time of maintaining DTP state. Blue colors represent control groups; Red colors represent MDR cancer cells. Mean with ± SD.
Figure 4Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. Representative dot plots showing IL-22 and IL-17A production in γδ T cells (A) and frequency of IL-22+ γδ T cells (B) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 6; 3 experiments, IMQ: n = 8-9; 3 experiments. Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. Representative dot plots showing IL-22 and IL-17A production in γδ T cells (C) and frequency of IL-22+ γδ T cells (D) following culture without (−) or with 40ng/ml recombinant IL-23 (n = 9-10, 3 experiments). Frequency of IL-22+ γδ T cells following culture with IL-1β, IL-23 or nothing. Each symbol represents one experiment. Open circles = Cre+ (each color represents a different culture condition).