EMBO/sd-character-level-ner huggingface dataset:
Figure 2Effect of TFL457, TFL482 and TFL541 on TrkB-FL levels. Cultures preincubated with TrkB-FL peptides or TMyc (25 µM, 30 min), followed by chronic NMDA treatment (2 h), were compared to cells without peptide. Levels of full-length (FL) TrkB were established with panTrkB, an antibody for the extracellular domain that also detects the truncated forms (tTrkB). Quantitation of peptide interference of TrkB-FL downregulation. Receptor levels were normalized to NSE and expressed relative to values obtained in cultures without peptide or NMDA. Means ± SEM are represented and analysis was performed by ANOVA test followed by post-hoc Tukey HSD test (*p = 0.02; n = 4). Effect of TFL457, TFL482 and TFL541 on neuronal viability after chronic excitotoxicity. Cultures were preincubated with peptides and treated with NMDA for 0-6 h as before. Values for each time point were represented relative to those of neurons incubated with the same peptide but no NMDA. Mean ± SEM of eleven (TFL457), three (TFL482) and seven (TFL541) independent experiments is given. Effect of TFL518 on TrkB-FL levels. Cultures incubated with TFL518 as before were compared to those treated with TMyc or TFL457. TFL518 effect on neuronal viability. Cultures were treated and analyzed as above. No significant differences were found for TFL518 while neuroprotection by TFL457 was confirmed Effect of TFL457 on neuronal viability after acute excitotoxicity. Cells were induced with NMDA (50 μM) and glycine (10 μM) for 1 h and, after agonists removal, culture proceeded for 20 h with DL-AP5 (200 μM) and TMyc or TFL457 (15 μM). Mean ± SEM relative to cultures incubated with TMyc but no NMDA is represented.
Figure 2Schematic diagram of the experiment. Representative confocal z-stack images of PSD95 (green)- and VGLUT1 (red)-positive excitatory (A) and Gephyrin (green)- and VGAT (magenta)-positive inhibitory (B) synapses in the CTX, HP, and IC (CTX: cortex, HP: hippocampus, and IC: inferior colliculus) of control and Cdc50a cKO mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm. Bar graphs showing the numbers of excitatory (C, PSD95 and VGLUT1) and inhibitory (D, Gephyrin and VGAT) synapses in the CTX, HP, and IC of control and Cdc50a cKO mice (n = 5 for each group, for excitatory synapses: n.s., not significant, for inhibitory synapses: CTX, P*** = 0.0001, P** = 0.0023, HP, P** = 0.0038, IC, P** = 0.0023, P**** < 0.0001). Representative confocal z-stack images of GABAA receptor α (red) and β (green) staining in the IC in control and Cdc50a cKO mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm. Representative current clamp traces from the IC of control and Cdc50a cKO mice; +100 pA (upper) and +400 pA (lower) currents were injected into control mice (left) and Cdc50a cKO mice (right) for 500 ms. Frequency-current (F-I) curve of the IC of control and Cdc50a cKO mice. The number of spikes per 500 ms was measured and averaged for each current (each, n = 9 and 13 cells, 300 pA: *P = 0.0203, 350 pA: *P = 0.0133, 400 pA **P = 0.0098, 450 pA: **P = 0.0072).
Figure 4(A-D) Ulk1 puncta formation was induced by etoposide in a PPM1D-dependent manner. The indicated MEFs were treated with etoposide (10 µM) with or without GSK2830371 (20 µM) for the indicated times, followed by immunostaining with an anti-Ulk1 antibody. Representative images are shown in (A). Ulk1 puncta are observed in etoposide-treated PPM1D+/+MEFs. (B-D) The proportion of cells with Ulk1 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05, **p < 0.01, NS: not significant. (E-I) Role of the dephosphorylation of Ulk1 at Ser637 on etoposide-induced Ulk1 puncta formation and Atg13 phosphorylation. Ulk1/Ulk2 DKO MEFs stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were treated with etoposide (10 µM) for the indicated times. (E, F) Cells were immunostained with an anti-Ulk1 antibody (E), and the population of cells with Ulk1 puncta was calculated (n > 100 cells in each experiment) (F). Data are shown as the mean  ±  SD (n = 3 experiments). *p < 0.05, **p < 0.01. (E-I) Role of the dephosphorylation of Ulk1 at Ser637 on etoposide-induced Ulk1 puncta formation and Atg13 phosphorylation. Ulk1/Ulk2 DKO MEFs stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were treated with etoposide (10 µM) for the indicated times. (G-I) Cell lysates were collected in a time-dependent manner, and Atg13 protein levels and its phosphorylation at Ser317 were examined by western blotting. Semiquantitative analyses are shown in Fig. EV3D. (J) EGFP-DFCP1-expressing PPM1D+/+MEFs were treated with etoposide (10 µM) for the indicated times, followed by immunostaining with an anti-Ulk1 antibody. Representative images of EGFP-DFCP1 (green; left), anti-Ulk1 (red; center), and a merged image (right) are shown. The nuclei were stained with Hoechst 33342 in the merged image. A magnified image of the area within the dashed square is also shown. Arrowheads indicate DFCP1 puncta localized close to Ulk1 puncta. (K, L) The role of Ulk1 dephosphorylation at Ser637 on etoposide-induced DFCP1 puncta formation. Ulk1/Ulk2 DKO MEFs stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were transfected with EGFP-DFCP1 and treated with etoposide (10 µM) for the indicated hours, followed by immunostaining with an anti-HA antibody. Representative merged images of EGFP-DFCP1 (green), anti-HA (red), and Hoechst 33342 (blue) are shown in (K). Arrowheads indicate DFCP1 puncta localized close to Ulk1 puncta. The number of DFCP1 puncta in each cell was calculated and is shown in (L) (n > 8 cells in each experiment). Red lines indicate the mean values. **p < 0.01, NS: not significant.
Figure 6A-F Confocal images of ddaC neurons expressing UAS-mCD8::GFP (Green channel), UAS-Nod-lacZ and immunostained for β-galactosidase at wL3 stages. Simultaneous knockdown of klp10A and msps (B) significantly restored Nod-lacZ localization in the dendrites, compared to msps and control RNAi (A). ddaC neurons with colchicine treatment (D) or with Kat-60 overexpression (F) exhibited perturbed Nod-lacZ distribution with highly enriched staining in the soma and decreased signals in the dendrites. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured. G-L Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. Simultaneous knockdown klp10A and msps (H) almost fully rescued the dendrite morphological defects and the pruning defects compared with the msps, control RNAi neurons. Wild type larvae fed with colchicine (J) showed pruning defects in comparison with those feed with DMSO (I). ddaC neurons with Kat-60 overexpression (L) exhibited abnormal dendrite arborization and pruning defects. Red arrowheads point to the ddaC somas. M, N Quantitative analysis of normalized Nod-lacZ intensity for 20 μm of major dendrite located 55 μm or 40 μm away from the soma respectively. O Percentages of ddaC neurons showing severing defects at 16 h APF. P Quantitative analysis of unpruned dendrite lengths at 16 h APF.
Figure 4CD1-nu/nu mice carrying PC9 xenografts were divided in groups (2-3 mice/group) and treated for seven days with different TKIs: erlotinib (50 mg/kg or 20 mg/kg), osimertinib (5 mg/kg) or afatinib (5 mg/kg), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, each at 0.1 mg/mouse/injection). when singly applied used erlotinib at 50 mg/kg. (A) Shown are fold changes in tumor volume at day 7 of treatment. The horizontal line indicates no change in tumor volume (fold change) between days 0 and 7 of treatment. CD1-nu/nu mice carrying PC9 xenografts were divided in groups (2-3 mice/group) and treated for seven days with different TKIs: erlotinib (50 mg/kg or 20 mg/kg), osimertinib (5 mg/kg) or afatinib (5 mg/kg), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, each at 0.1 mg/mouse/injection). when singly applied used erlotinib at 50 mg/kg. (B) After treatment, all mice were sacrificed and tumors extracted. Protein extracts were resolved by means of electrophoresis and transfer to nitrocellulose membranes, which were later incubated overnight with the indicated antibodies. This was followed by incubation with peroxidase-conjugated secondary antibodies. Signals were detected using ChemidocTM (from Bio-Rad), quantified and normalized to the signals of GAPDH or tubulin (numbers shown below each lane).
Figure 1(A) Representative micro-CT images and H&E staining of trabecular bone from the femoral metaphysis of 6-week-old male Sp7-Cre;Cdc20f/f and littermate control mice. Scale bar, 500 μm. (B) Histomorphometric analyses of 6-week-old male femurs. (n=6) (C) Representative micro-CT images and H&E staining of trabecular bone from the femoral metaphysis of 6-week-old female Sp7-Cre;Cdc20f/f and littermate control mice. Scale bar, 500 μm. (D) Histomorphometric analyses of 6-week-old female femurs. (n=6) (E) Representative micro-CT images of tibial cortical bone defects in Sp7-Cre;Cdc20f/f and littermate control mice. The green lines show the position of the original defect position. The green circle represents the regenerated bone. Scale bar, 500 μm. (F) Histomorphometric analysis of the regenerated bone in tibial cortical gaps. (n=5)
Double perturbations reveal patterns of interactions between Hoxb8 and other transcription factors.Figure 4A Experimental design: Hoxb8-FL were transduced with sgRNAs targeting the indicated TF (or a control sgRNA) and subjected to β-estradiol withdrawal (switching off Hoxb8 ectopic expression - Hoxb8*) or cultured in normal conditions (control). B A two-factor linear model with interaction used to fit the data. Observed expression (Y) is modelled as a sum of expression in control cells (β0), effect of perturbation 1 (binary factor X1 and coefficient β1), effect of perturbation 2 (X2 and β2), their interaction (X1X2 and β3) and the error term (∑). The interaction term can be interpreted as the difference between the expression changes in the double-perturbed cells and the sum of coefficients of β1 and β2. C Binary combinations of directions in observed expression changes for perturbation 1, perturbation 2 and the interaction term grouped into four general classes. Based on classification implemented in (Dixit et al, 2016). D Changes in expression for genes co-regulated by separate Cebpa/Meis1/Spi1 and Hoxb8 perturbations (FDR <0.1 and |log2(FoldChange)| > 0.2). The interaction row indicates changes beyond simple additive effect (white = additive effect). Each gene was annotated with an interaction class (int. class) as explained in (C), using the |log2(FoldChange)| > 0.2) threshold to assign change direction. E Fractions of genes in each interaction class showed in (D).
Figure 9Tau coacervation with RNA (1) - or other polyanions - without PEG produces non-percolating condensates that are instable at physiological ion concentrations. Phosphorylation (pTau) inhibits coacervation of Tau. Crowding-induced condensates (2) are stable at physiological salt concentrations and percolate into gel-like condensates within 24 h. Tau:PEG condensates promote MT growth but disassemble during the polymerization process. Condensation of Tau with RNA in the presence of molecular crowding (3), a condition that likely exists in the cytosol, produces condensates that are stable at physiological ion concentrations and also percolate into gel-like condensates. Tau:PEG:RNA condensates strongly promote MT bundling, and they persist as condensed phases attached to MT bundles, thereby inducing a distinct spatial organizing of MT bundles. Tau:PEG:RNA condensates can function as 'liquid matrix' that can host different otherwise not compatible Tau binding partners, such as RNA and tubulin. Regardless of their physiochemistry and origin, all condensates induce the formation of SDS-stable Tau dimers and oligomers with time, and efficiently induce Tau aggregation in sensor cell and neuron models. Tau condensates induce spherical accumulations in the nucleus (NUC), large aggregates in the cytosol (CYT) adjacent to the nucleus, and small granular Tau condensates at the nuclear envelope (NE), which in part colocalize with nucleoporins and impair the nucleocytoplasmic transport. In contrast, heparin-induced Tau aggregates mostly seed CYT accumulations.
Figure 3(A) Poly(GP) levels were measured by MSD immunoassay in C9orf72 patient iPSC-motor neurons treated for 7 days with 8-16 µM of the G4C2 repeat G-Q binding small molecules DB1247, DB1247 and DB1273. Treatment with DB1246 or DB1273 leads to a significant reduction in poly(GP) levels relative to vehicle-treated controls. Data are shown as the mean and SEM of 3 independent C9orf72 iPSC lines, with 1-6 differentiations per line. *p<0.05, **p<0.01, *** p<0.001, one sample two-tailed t-test versus normalised control. p=0.0068 (DB1246, 12 µm), p=0.0417 (DB1246, 16 µm), p=0.0196 (DB1247, 8 µm), p=0.0002 (DB1273, 8 µm), p=0.0194 (DB1273, 12 µm), p=0.0062 (DB1273, 16 µm) (B) C9orf72 patient iPSC-motor neurons were treated for 7 days with 16 µM of each small molecule and the expression levels of MCM2 and the three C9orf72 transcript variants (V1-V3) measured by quantitative RT-PCR. Data are shown as the mean and s.d. of 3 independent iPSC-motor neuron lines (one induction per line) relative to vehicle (DMSO) treated controls. No significant changes in gene expression were observed, one way ANOVA, Dunnett's post hoc test, p>0.05. (C) XTT cell death assay for C9orf72 iPSC-motor neurons treated for 7 days with 0 - 40 µM of DB1246, DB1247, DB1273 or cisplatin as a positive control. Data are shown as the mean and SEM of two independent iPSC-motor neuron lines. Toxicity is only observed at the highest dose of 40 µM and not at the penultimate dose of 20 µM. (D) Drosophila first instar larvae expressing 36 G4C2 repeats were placed on food containing either vehicle or 1 mM DB1273, and the number reaching the pupal stage of development counted after 7 days. DB1273 treatment (n=1200 flies) significantly improves survival compared to vehicle (n=1150 flies), p=0.0320, chi-squared test. Data are shown as proportion reaching the pupal stage, with numbers within each group indicated on the bars. Genotype was w1118; (GGGGCC) 36/+; daGAL4/+ (daGAL4>36R). (E) Drosophila first instar larvae ubiquitously expressing 36 G4C2 repeats (36R) were treated with vehicle or 1 mM DB1273 for 5 days, until the third instar stage (L3), and poly(GR) measured by MSD immunoassay. Poly(GR) was also measured in control (w1118) larvae that do not express G4C2 repeats. DB1273 treatment significantly reduced poly(GR) expression. n=8 for 36R groups, n=7 for control group. **p<0.01, *** p<0.001, one way ANOVA with Dunnett's post hoc test. p=0.0001 (w1118 versus 36R 0 mM larvae), p=0.0019 (0 mM versus 1 mM 36R larvae). Genotypes were w1118; ; and w1118; (GGGGCC) 36/+; daGAL4/+ (daGAL4>36R).
XBP-1S expression in clinical prostate cancer specimens.Figure 7XBP-1S expression was determined by IHC in two different cohorts of human prostatectomy samples.Representative pictures of benign and tumor samples. Tissue microarrays (Wang et al, 2010) containing 25 benign and 283 tumor samples were stained with a XBP-1S specific antiserum and scored by a pathologist. The P-value indicates the difference between XBP-1S staining (strong and moderate) in normal vs cancer cells using Mann-Whitney test. XBP-1S expression was determined by IHC of a neoadjuvant hormone therapy (NHT) tissue microarray containing samples from hormone naïve (untreated) (n = 25), patients that received NHT for 1-6 months (n = 33), and patients that received NHT for 7-12 months (n = 50), as indicated. Representative images are shown. Scale bars: 100 μm.Quantitative presentation of the data from (C). *P = 0.006 in unpaired Student's t-test. A model for AR regulation of UPR in PCa cells: Liganded AR activates the IRE1α pathway and coordinately inhibits the PERK arm of the UPR. In addition, AR inhibits the proapoptotic JNK pathway that may be activated by IRE1α or other pathways. The end result of these AR effects is PCa cellproliferation and survival. An arrow with a solid line indicates direct promotion. An arrow with dashed line indicates indirect/unexplored interactions.
Figure 2(A) Western blotting analysis of p53 expression in various p53/TP53-WT cell lines incubated with the DNA-intercalating agent doxorubicin (DOXO, 0.2 µg/ml for 6 hours) to induce p53 stabilization (n = 3 biological replicates). (B, C) Western blotting analysis confirming NF1 upregulation and p-FAK1 downregulation in (B) DOXO-treated PDKN1 cells and (C) DOXO-treated PDKN2 cells transduced with PGK-NF1 (n = 3 biological replicates). (D) Western blotting analysis confirming NF1 downregulation and p-FAK1 upregulation in DOXO-treated SW1573 cells transduced with sgNf1.3 (n = 3 biological replicates). (E, F) Western blotting analysis confirming Nf1 downregulation and p-Fak1 upregulation in (E) DOXO-treated LKR10 and (F) DOXO-treated LKR13 clones transduced with sgNf1.3 (n = 3 biological replicates). (G) FAK1 target gene expression in DOXO-treated PDKN1 cells transduced with PGK-control or PGK-NF1 (n = 3 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs). (H, I) Subcutaneous tumor volumes of DOXO-treated PDKN1 and PDKN2 cells transduced with PGK-control or PGK-NF1 (n=18 each). (J-L) Subcutaneous tumor volumes of (J) SW1573, (K) LKR10, and (L) LKR13 cells transfected with sgTom or sgNf1.3 (n=18 each). (M) Depictions and quantitation of KrasG12D/+; p53+/+ (K-only) autochthonous tumor burden (total tumor area/total lung area) in pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean. (N) Analysis of tumor grades in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For bar charts, data presented as means with error bars representing standard deviations (SDs). (O) Quantification of Ki-67-positive nuclei per mm2 in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.
Figure 4(A) Expression changes of different classes of TEs in Tet3-KD and Tet-TKD. Shown are log2-fold changes of KD embryos compared to control 2-cell embryos of different repetitive elements subdivided in classes of TEs. Indicated p-values were calculated using a paired two-tailed t-test (*** = p < 0.001, **** = p <0.0001, data are represented as medium smoothed violin plots with indicated median and quartiles as dotted lines). (B-C) Expression levels of (B) maternally expressed ERV3-LTRs and (C) 2-cell embryo specific ERV3-LTRs in control, Tet3-KD and Tet-TKD 2-cell embryos (p-values were calculated using DEseq2; * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, data are represented as mean ± SD, dots represent single embryos). (D) Volcano plots showing expression changes of genes in Tet3-KD and Tet-TKD 2-cell embryos. Chimeric genes, as defined by the presence of a nearby MERVL element and upregulation in 2-cell embryos, are indicated (see (Macfarlan et al., 2012)). Dashed lines represent the cut off for differential expression: |log2FC| > 0.7 and padj < 0.05. (E) Expression levels of important candidate factors in control, Tet3-KD and Tet-TKD 2-cell embryos, implicated in the generation of 2C-like cells and as important regulators of EGA (p-values were calculated using DEseq2; * = p < 0.05, ** = p < 0.01, **** = p < 0.0001, data are represented as mean ± SD, dots represent single embryos).
Conceptual Summary Impairment of EXOSC10 with arginine-rich DPR compromises repeat RNA degradation through the RNA exosome complex.Figure 9Impairment of EXOSC10 with arginine-rich DPR compromises repeat RNA degradation through the RNA exosome complex. This accelerates repeat RNA accumulation (as RNA foci) and RAN translation dependent DPR production. The produced DPR in turn compromises EXOSC10/the RNA exosome complex and thus accelerates the pathological process of C9orf72 FTLD/ALS.
Figure 1(A) Workflow showing phosphopeptide enrichment strategy. Brain regions were dissected from mice and then processed into proteolytic peptide digests and labeled for quantification with TMT. Phosphopeptides were then enriched and analyzed by liquid chromatography followed by tandem-mass spectrometry. Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from CK-p25 animal mice. Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte. Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from 5XFAD animal mice. Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte. (D) Phosphosite enrichment analysis of phosphopeptides from CK-p25, 5XFAD, and Tau mice. Circle colors indicate normalized enrichment scores (NES) and sizes indicate the number of observed sites in each substrate phospho-set within each mouse model. Kinases enriched in at least one model are shown. Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte. (E) Heatmap showing enriched hippocampus phosphotyrosine peptides from 6 month old Tau P301S animals. (F-G) Relative abundances across all tissues for phosphotyrosine peptides: (F) SVyTEIK (Siglec-F pY561); (G) LyDFVK (Inpp5d pY868). Bars indicate mean ± 95% confidence interval (CI); n = 3-8 samples; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.