EMBO/sd-character-level-ner huggingface dataset:
Figure 10A Co-immunostaining of PTPσ (blue), NB-3 (green), and BDA (red) at spinal lesion sites of NB-3+/+ (A1) and NB-3−/− (A6) mice. (A2-A5 and A7-A10) High-magnification images of boxed areas in (A1 and A6) showing PTPσ expression at the terminations of BDA-labeled corticospinal axons, respectively. Images in (A2 and A7) are presented as single-channel fluorescence images in (A3-A5 and A8-A10), respectively.B Quantification of fluorescence intensity of PTPσ at BDA-labeled axonal terminations of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05; one-way ANOVA followed by Bonferroni post-test. n = 12 mice per group. C Co-immunostaining of PTPσ and NB-3 in the FG-labeled corticospinal neurons (arrowheads) of NB-3+/+ and NB-3−/− mice 14 days post-injury (dpi). Insets, only the signals from PTPσ and FG in NB-3+/+ corticospinal neurons (red arrowheads) were visualized.D Quantification of fluorescence intensity of PTPσ in (C). *p < 0.05; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified. E Co-immunoprecipitation of NB-3 and PTPσ from sensorimotor cortex lysates of NB-3+/+mice after SCI.F Quantification of immunoprecipitated PTPσ by NB-3 in (E). **p < 0.01; one sample t-test. Data were analyzed from 3 independent experiments each including 15 mice per group. G PTPσ expression in NB-3+/+ and NB-3−/− pyramidal neurons when co-culturing with NB-3-expressing astrocytes (dashed lines and asterisks) or astrocytes that didn't express NB-3 (dashed lines). Upper inset, high-magnification view of the soma (white arrowhead). Lower inset, high-magnification view of the axon tip (green arrowhead).H Quantification of fluorescence intensity of PTPσ in (G). n.s., not significant, and *p < 0.05; one-way ANOVA followed by Bonferroni post-test. I Expression of PTPσ, p-Akt, p-mTOR, and p-S6 (S240/244) in pyramidal neurons that expressed PTPσ shRNA-GFP. A control plasmid expressing GFP was transfected as the negative control (nc).J Quantification of fluorescence intensity of PTPσ, p-Akt, p-mTOR, and p-S6 (S240/244) in (I). *p < 0.05; one sample t-test. The intensities of more than 300 pyramidal neurons from 4 independent experiments in each group in (H) and (J) were quantified, respectively. K-M Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV-nc-GFP (K) and LV-PTPσ shRNA-GFP (L) into the sensorimotor cortex. High-magnification image (M) of the area in (L) (asterisk). The arrows indicate the lesion sites; the asterisk indicates the lesion epicenter.N Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (K and L). *p < 0.01; two-way ANOVA followed by Fisher's LSD. n = 8 mice in (K), and n = 10 mice in (L). O A model illustrating the signal transduction between scar-forming cells and post-traumatic axons mediated by trans-homophilic NB-3 interactions.
Figure 7A-C Tethering assay using the R-Luc-6xMS2-A95-MALAT1 reporters containing the indicated 5'-UTRs and MS2-HA-tagged DDX6 or a DDX6 mutant (Mut1) that does not bind to NOT1 in HEK293T cells. R-Luc activity and mRNA levels were normalized to those of the F-Luc control and analyzed as described in Fig 2C,D. D Immunoprecipitation assay showing the interaction of DDX6 or DDX6 Mut1 with GFP-NOT1 in HEK293T cells. Inputs (1% for the HA-tagged proteins and 3% for the GFP-tagged proteins) and immunoprecipitates (20% and 15%, respectively) were analyzed by western blotting using anti-HA and anti-GFP antibodies. Endogenous CNOT2 and CNOT3 (input 0.5% and IP 10%) were detected using specific antibodies (Appendix Table S4). E Tethering assay using the F-Luc-HhR-A95-C7-Hhr reporters containing an 8-nt or a 109-nt 5'-UTR and λN-HA-Me31B in S2 cells. F-Luc activity and mRNA levels were analyzed as described in Fig 2A and B. F,G Tethering assay using the R-Luc-6xMS2-A95-MALAT1 reporter and MS2-DDX6 in HEK293T. In panel (F) luciferase activity was analyzed as described in Fig 2C. The association of the R-Luc reporter and the F-Luc control with polysomes was analyzed by sedimentation through a sucrose gradient. Panel (G) shows the amount of the R-Luc mRNA in each fraction normalized to the total amount across all fractions in cells expressing MS2 (black curve) or MS2-DDX6 (red curve). Lower panels show representative northern blots. The data corresponding to the F-Luc control and the corresponding ribosomal RNA profiles are shown in Fig EV4E and F.P
WIPI2 Binds and Colocalizes with the Atg12–5-16L1 Complex.Figure 1(A) HEK293A cell lines stably expressing GFP, GFP-WIPI1a, and GFP-WIPI2b were incubated in full medium (F) or starvation medium (EBSS) (S) for 2 hr before being used for GFP-TRAP pull-down. (B) WIPI2 was immunoprecipitated from HEK293A cells treated with DSP, at the indicated concentrations, before immunoblotting. (C) WT or Atg16L1Δ/Δ MEFs in fed medium or starved for 2 hr in EBSS were fixed and labeled with an anti-WIPI2 antibody. Scale bars, 10 μm.(D) WIPI2 puncta in (C) were counted, and a statistical analysis of WIPI2 puncta was performed using an unpaired Student's t test. ∗p < 0.05. SEM for n = 3. (E-H) CLEM (correlative light and electron microscopy) of endogenous WIPI2 in Atg16L1Δ/Δ MEFs.(E) Merged phase and confocal section of Atg16L1Δ/Δ cells labeled with anti-WIPI2 antibody. Scale bar, 10 μm.(F) Low-magnification TEM of cell in (E). Scale bar 10μm.(G and H) High magnification of boxed regions in (E) and (F). Top box and bottom box indicate panels (G) and (H), respectively. Arrows indicate open phagophores. M, mitochondria; ER, endoplasmic reticulum. Scale bars, 1 μm (G) and 0.5 μm (H). (I and J) 2GL9 cells (GFP-LC3 HEK293 cells; Chan et al., 2007) were starved for 2 hr before visualization using indicated antibodies. Scale bars, 10 μm. (K) Live-cell imaging demonstrates that Atg16L1 translocates to WIPI2b-positive puncta in starvation. HEK293 cells expressing GFP-WIPI2b and mCherry-Atg16L1 were starved in EBSS and imaged every 2 s using a spinning disk microscope. See also Figure S1 and Movie S1.
Figure 1Panel A shows R2 and AIC for different regression models, using either only amyloid plaque density ("P"), only tangle density ("T"), both plaque and tangle density ("P+T"), or both plaque and tangle density including their interaction term ("PxT"). All models included age and sex as covariates. The panel shows adjusted R2, together with AIC (above the bars) for each model. We compared R2 between the models using a bootstrap procedure (N=1000 iterations), which showed that the R2 for the "PxT" model was marginally higher than for the "P+T" model (∆R2=0.027, 95% CI -0.0034-0.087), and significantly higher than for the "T" model (∆R2=0.12, 95% CI 0.04-0.24) and the "P" model (∆R2=0.18, 95% CI 0.055-0.31). Panel B shows associations between plasma P-tau217 and plaque density (stratified by tertiles [T1-3] of tangle density). Panel C shows associations between plasma P-tau217 and tangle count (stratified by tertiles [T1-3] of plaque density).
Figure 2(A,B) ELISA screen of the lectin-Fc library against full-length trimeric SARS-CoV-2 Spike (A) or monomeric RBD (B). Results are shown as mean OD values of 2 replicates normalized against a BSA control and ranked by value. Lectin-Fc fusion proteins with a normalized OD > 0.5 in either (A) or (B) are indicated in both panels. (C) Lectin-Fc and human ACE2-mIgG1 Fc-fusion protein (hACE2) binding to untreated or heat-denatured full-length SARS-CoV-2 Spike by ELISA. hACE2-mIgG1 was used as control for complete denaturation of Spike protein. Results are shown as mean OD values ± SD normalized to the BSA control (technical replicates, N=3). (D) Lectin-Fc binding to full-length SARS-CoV-2 Spike with or without de-N-glycosylation by PNGase F. "PNGase F only" denotes wells that were not coated with the Spike protein. Results are shown as mean OD values ± SD normalized to BSA controls (technical replicates, N=3). (E,F) Surface plasmon resonance (SPR) analysis with immobilized full-length trimeric Spike, probed with various concentrations of Clec4g-Fc (E) and CD209c-Fc (F).
Figure 5A, B. Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of exogenous hFis1. WT 293T cells stably expressing mitoGFP or mitoRFP were co-cultured, subsequently transfected with empty vector (control) or with Myc-hFis1 as indicated and then treated with PEG for cell fusion. Mitochondrial fusion is indicated by co-localization of mitoGFP and mitoRFP (i.e. yellow mitochondria). Left panel: empty vector transfected; right panel: Myc-hFis1transfected. Insets represent magnification of the boxed areas in upper panel (A). Quantitative analysis of the extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient (PCC) in three independent experiments for each condition and data summarized in (B). C, D. Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of exogenous hFis1. WT 293T cells co-transfected with mito-PAGFP (0.5 µg), mito-DsRed (0.2 µg), and either empty vector (0.5 µg, the upper panel) or hFis1 (0.5 µg, the lower panel) were photoactivated in a small region of interest (ROI) (white circle, 3 µm diameter) in preactivation images of mitochondria seen by mito-DsRed (red). After photoactivation, sequential Z-stack images with photoactivated GFP (green) and mitochondrial marker (mito-DsRed) were collected at indicated time points using series of Z-sections from the top to the cell bottom with intervals between sections set to 0.5-0.75 μm (C). Mitochondrial fusion was quantified by analyzing changes in fluorescence intensity of photoactivated mito-PAGFP in ROIs at 40 sec, 15, 30, 45 min after photoactivation. The dilution rates (percentage) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation (D). E, F. Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of endogenous hFis1. Both WT 293T cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA (control siRNA) or hFis1-siRNA as indicated and then co-cultured and fused using PEG treatment. Left panel: control siRNA transfected; right panel: hFis1-siRNA transfected (E). Quantitative analysis of the mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient in three independent experiments and data summarized in (F). G, H. Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of endogenous hFis1. Scrambled siRNA or hFis1 siRNA treated cells were co-transfected with mito-PAGFP and mito-DsRed and were subsequently photoactivated (G) as described in (C). Mitochondrial fusion was quantified (H) as described in (D).
Generation of iPSC-derived NCC.Figure 2A. Schematic of the differentiation protocol timeline. Maintenance Medium (MM) = iPSC medium (StemFlex with 1x penicillin/streptomycin), NCC differentiation medium = DMEM-F12, 10% fetal bovine serum, 1 mM sodium pyruvate, 1 mM penicillin/streptomycin, 1 mM non-essential amino acids, 110 µM 2-mercaptoethanol, 10 ng/mL epidermal growth factor. B. Images of iPSC and iPSC-derived NCC at Days 0, 14, and passage 4 following differentiation. Scale bars: 400 µm (Day 0), 200µm (Day 14, passage 4).
a) Body weight was measured in 5-month-old and 12-month-old female Pgam5 WT and KO mice.f4(a) Body weight was measured in 5-month-old and 12-month-old female Pgam5 WT and KO mice. Behavioral measurements on 12-month-old female WT and KO mice (WT n=10, KO n=8) were as follows: (b) Open-field tests including time spent mobile, time spent immobile, whole area distance and centre area distance; Behavioral measurements on 12-month-old female WT and KO mice (WT n=10, KO n=8) were as follows: (c) Gait length, gait frequency in steps/ss and step angle; (d) rearing test, (e) akinesia test and (f) ladder climb test and (g) vertical pole test. T-turn indicates the time mice spent to turn their bodies 180 degrees from the head up start position before descending the pole. T-down indicates the time mice spent to descend to the bottom of pole. Graphs show the mean and s.d. values and were analysed by the student's t-test, *P0.05, **P0.01, ***P0.001, NS, not significant.
Figure 5Representation of the analysis scheme for axonal transport of mitochondria in Ccp1flox/floxCcp6flox/flox neurons. Hippocampal neurons from E17.5 embryos were cultured and transduced (on DIV0) with lentivirus expressing either GFP (control) or GFP-2A-cre (inducing conversion to Ccp1-/-Ccp6-/-). At DIV4, live neurons were stained with MitoTrackerTM and mitochondria were imaged in GFP-positive cells for 1 min with a spinning-disk microscope. Mitochondria movements were plotted in kymographs for all the cells analyzed in each set and broken down into single runs without change in direction or speed. Anterograde and retrograde runs were color-coded in green and red, respectively, while immotile events are shown in blue. Immunoblot of cell extracts from Ccp1flox/floxCcp6flox/flox hippocampal neurons without, or after transduction with lentivirus expressing either GFP or GFP-2A-cre after 4 days in culture. Note the specific increase of polyglutamylation (polyE) in neurons transduced with GFP-2A-cre lentivirus. All kymographs of one representative set of experiments. Mitochondria movements (Movie S1) were plotted in kymographs and analyzed as described in (A). Scale bars: 60 sec, 20 µm. Analyses of (D) run length and (E) speed distribution, represented as scatter dot plots with each point representing a single run extracted from the kymographs shown in (C). Each group represents the pool of measurement of multiple neurons made in the experiment. The black line indicates the median of the distribution with whiskers at interquartile range. Numerical value of the medians is indicated below each plot. (F) Overall motility of mitochondria represented as fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s. Values (below plots) from one set of experiment (C) show a decrease of about 50% in Ccp1-/-Ccp6-/- conditions, while (G) mitochondria density is unaltered. (H-K) Statistical analyses of mitochondria transport parameters of experiments shown in (D-G; Fig. EV4A-D). Each bar represents the mean (±SEM) of medians of single experiments for run length (H) and speed (I) distributions, and average (±SEM) mitochondria motility (J) and density (K) of all seven experimental sets. Values of fold change between control and Ccp1-/-Ccp6-/- conditions are indicated below the graphs. Green bars are for anterograde, and red for retrograde transport events. Significance was tested using unpaired t-test. Average run length (H) as well as speed (I) showed no significant differences in these two parameters between control and Ccp1-/-Ccp6-/- conditions. (J) The average of overall motility reveals an about 50% decrease in overall mitochondria motility, while mitochondria density (K) is unaltered between control and Ccp1-/-Ccp6-/- conditions.
Figure 1A HeLa cells expressing LAMP1-RFP were treated with vehicle alone (control) or LLOMe for 1 h to induce lysosome damage. Cells were fixed 2 h after washout, stained with p62 and galectin-3 (Gal3) antibodies, and analyzed by confocal microscopy. B p97 translocates to ruptured lysosomes. Cells co-expressing LAMP1-RFP and GFP-p97 were treated as in (A) and stained with the pan-ubiquitin antibody FK2 (Ub). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD). C Immunodetection of endogenous p97 after LLOMe treatment as in (A). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD). D Clearance of damaged lysosomes depends on p97. Cells were transfected with control (Ctrl) or p97 siRNA oligos, fixed at indicated time points after LLOMe washout and stained for endogenous Gal3. Quantification of the number of Gal3-positive vesicles per cell. Data represent mean ± SD of four independent experiments with ≥30 cells quantified per condition (one-sided Welch's unequal variances t-tests comparing the siCtrl with sip97). E Cells were chased after LLOMe treatment for 12 h with vehicle alone (DMSO), bortezomib (Btz, 10 nM), NH4Cl (20 mM) or the p97 inhibitor NMS-873 (5 µM), and Gal3 vesicles quantified. Data represent mean ± SEM of three independent experiments with ≥30 cells quantified per condition (one-sided Welch's t-tests). F p97 is essential for cell survival after lysosome rupture. Cells were transfected with control (Ctrl), p97 or SEL1L siRNA oligos and treated with indicated concentrations of LLOMe for 12 h. Cell viability was measured using the MTS assay. Data represent mean ± SD of three independent experiments. G Taufibrils are endocytosed and induce lysosomal damage. Taufibrils were generated in vitro, fluorescently labelled by Dylight488 and fragmented by sonication (488-labelled tau). HeLa cells expressing mCherry-Gal3 were incubated with 300 nM taufibrils for 24 h. Arrows indicate compartments that contain 488-labelled tau and are positive for mCherry-Gal3 indicating lysosome rupture. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD). H Accumulation of tau-induced lysosomal damage upon p97 depletion. Cells transfected with control (Ctrl) or p97 siRNA oligos were treated as in (G). Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD). * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Scale bars, 10 µm.
SV2 is specifically reduced in spinal cord lysates of symptomatic transgenic GA149-CFP mice Spinal cord lysates were generated from fresh frozen tissue of 20-month old mice either expressing GA149-CFP under the Thy1 promoter (Tgx GA149) or wild type controls.Figure 9Spinal cord lysates were generated from fresh frozen tissue of 20-month old mice either expressing GA149-CFP under the Thy1 promoter (Tgx GA149) or wild type controls. Lysates were immunoblotted for SV2 (A) Total protein levels were determined by imaging the Mini-PROTEAN TGX stain-free gel prior to transfer. Quantification of band intensities normalized to total protein loading revealed a significant reduction in SV2 protein levels in the GA-expressing cohort compared with wild type animals, Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 4 animals per group were examined. Spinal cord lysates were generated from fresh frozen tissue of 20-month old mice either expressing GA149-CFP under the Thy1 promoter (Tgx GA149) or wild type controls. Lysates were immunoblotted for synaptophysin (B) Total protein levels were determined by imaging the Mini-PROTEAN TGX stain-free gel prior to transfer. synaptophysin levels remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 4 animals per group were examined. Spinal cord lysates were generated from fresh frozen tissue of 20-month old mice either expressing GA149-CFP under the Thy1 promoter (Tgx GA149) or wild type controls. Lysates were immunoblotted for PSD-95 (C). Total protein levels were determined by imaging the Mini-PROTEAN TGX stain-free gel prior to transfer. PSD-95 levels remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 4 animals per group were examined.
Figure 6A, Venn diagram showing a comparison between TSN2_NS, TSN2_HS and TSN2_NaCl protein pools. B, Co-immunoprecipitation (Co-Ip) of TSN and RBP47 in protein extracts prepared from 10-d-old Arabidopsis seedlings expressing Pro35S:GFP-RBP47 and grown under no stress (NS), HS (39ºC for 60 min) or salt (NaCl) stress (150 mM NaCl for 60 min) conditions. The GFP-expressing line was used as a negative control. Endogenous TSN (107 kD) was detected in total fractions (Input) and fractions co-immunoprecipitated (Co-Ip) with RBP47 but not with free GFP in all three conditions. Input and Co-Ip fractions were analyzed by immunoblotting using α-TSN and α-GFP. C, Localization of GFP-tagged proteins in root cells of 5-d-old Arabidopsis seedlings expressing Pro35S:GFP-RBP47, Pro35S:GFP-UBP1, Pro35S:GFP-TCTP and ProUBQ:GFP-SnRK1α2. The seedlings were grown under 23°C (NS), incubated at 39°C for 60 min (HS) or treated with 200 mM NaCl at 23°C for 60 min (NaCl). Scale bars = 10 μm
Biochemical characterization of RBD-specific Nbs.Figure 2A Amino acid sequences of the complementarity determining region (CDR) 3 from unique Nbs selected after two rounds of biopanning are listed (upper panel). Phylogenetic tree based on a ClustalW alignment of the Nb sequences is shown (lower panel). B Recombinant expression and purification of Nbs using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Coomassie staining of 2 µg of purified Nbs is shown. C For biolayer interferometry (BLI) based affinity measurements, biotinylated RBD was immobilized on streptavidin biosensors. Kinetic measurements were performed by using four concentrations of purified Nbs ranging from 15.6 nM - 2 µM. As an example, the sensogram of NM1230 at indicated concentrations is shown (upper panel). The table summarizes affinities (KD), association (kon) and dissociation constants (koff) determined for individual Nbs (lower panel).
Figure 2Groups of Jpx#9 sequence variants in NucLibC. Just the indicated bases from the 3' of the tile are shown. The WT sequence is on top and few representative variants out of the 3,810 variants in NucLibC are shown. The altered regions are in bold and underlined. For each sequence the oligo name is shown and it includes the position(s) that were changed and the details of the change. Variants are grouped by the type of change made, and the region in which changes were introduced is shaded. Nuc/Cyto (top) and IP/Input (bottom) ratios for single mutations in NucLibC. The WT sequence is shown at the bottom. Each point is a sequence variant, color-coded based on the base that was introduced. Shaded regions correspond to regions where mutations affect binding but not function (purple), affect function and binding (ivory), and affect function with small effects on binding (green). Asterisks indicate positions where the Nuc/Cyto ratios of the mutated tiles are significantly different than those of the WT tile (Wilcoxon two-sided rank-sum test P<0.05) Color-coded Nuc/Cyto ratios of Jpx#9 variants where the indicated number of bases were deleted (and moved to the 5' of the sequence), starting from the indicated position (aligned to the sequence in (B)). Deletions to the right of the red line affect the SIRLOIN core region. Deletions to the right of the black line include the entire SIRLOIN core region (the deleted region is relocated to the 5' of the tile). Asterisks indicate positions where the Nuc/Cyto ratios of the tile with the specific deletion are significantly different than those of the WT tile (Wilcoxon two-sided rank-sum test P<0.05). The histogram on the right shows distribution of the presented Nuc/Cyto ratios.