EMBO/sd-character-level-ner huggingface dataset:
a) Fbxo7 relocates from the cytosolic to the mitochondrial fractions of HEK293T cells treated with CCCP (10 μM).figf2(a) Fbxo7 relocates from the cytosolic to the mitochondrial fractions of HEK293T cells treated with CCCP (10 μM). IB, immunoblot. (b) Fbxo7 levels are increased in Flag-Parkin complexes immunoprecipitated (IP) from the mitochondrial fraction of HEK293T cells transfected with Flag-Parkin and untagged Fbxo7 following 1 or 3 h treatment with CCCP (10 µM). (c) Parkin localization at the mitochondria was assessed by immunocytochemistry in SH-SY5Y cells transfected with Flag-Parkin plus scrambled (scr) or Fbxo7 siRNA, following 1 or 3 h treatment with CCCP (10 μM). Cells were scored visually for the colocalization of Flag-Parkin with HtrA2, a mitochondrial marker. Images are displayed for cells transfected as indicated, following 0 or 3 h CCCP treatment. For corresponding images at 1 h treatment, see Supplementary Figure 2c. Nuclei (blue) were stained with DAPI. Scale bars, 10 μm. (d) Loss of Flag-Parkin translocation upon Fbxo7 silencing is rescued by WT and R378G Fbxo7, but not by T22M Fbxo7, by R498X Fbxo7 or by Fbxo7 in which the mitochondrial targeting sequence is mutated (mt-MTS). For c,d, histograms indicate the percentage of cells in which Parkin localized to the mitochondria. Data are presented as mean of three experiments ± s.e.m. *P 0.05, **P 0.01 compared to cells transfected with Flag-Parkin plus Fbxo7 siRNA. (e) T7-tagged WT Fbxo7 localizes to both cytosolic (C) and mitochondrial (M) fractions of transfected HEK293T cells, but MTS mutant (mt-MTS) Fbxo7 localizes only to the cytosolic fraction. CxVα and PDHE1α are mitochondrial markers; GAPDH is a cytosolic marker. All western blots are representative of experiments performed at least three times. Full-length blots are presented in Supplementary Figure 9.
Fig.Figure 4Schematic of the xenograft mouse model for MB treatment. C57BL/6J mice were inoculated with EG7-L1 cells (2 × 106 cells, s.c.) on the right flank on day 1, followed by injection (2 × 106 cells, i.v.) of CD45.1+ CTL on day 3 and day 6 respectively. The mice were randomized into 3 groups (n = 5) and treated with vehicle, MB (20 mg/kg/day, i.g.), aPD1 (10 mg/kg, every other day, i.p.) served as positive control. Tumor size was measured through calipering every other day. EG7-L1: EG7 overexpressing PD-L1.   Tumor growth curve for (A) was shown as mean ±s.e.m.   Representative image of the tumor on day 10 mentioned in (A).   Tumor weight on day 10 (n=5) mentioned in (A).   Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Immunohistochemical analysis of cell proliferation marker ki-67 and CD8 in tumor nodules on day 10 (n=5) Statistical results of (E). Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Expression of Perforin, GZMB and IL-2 by CD8+ CD45.1+ TILs were analyzed by flow cytometry.   Statistical results of (G).   Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Schematic of EGFR L858R mice treated with MB.   Tumor burdens recorded through Computed tomography (CT) scanning for EGFR L858R mice (n=5, lung section = 3). Mice were treated with MB (20 mg/kg/day, i.g.) for 1 week, followed by imaged with CT again to record tumor burden.   Statistical results of (J).   Haematoxylin and Eosin staining of lung section of EGFR L858R mice (n = 5, lung section = 3) in (J).   Statistical results of (L).   Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 6Uptake of synaptotagmin-1 antibody during high K+ stimulation. Neurons expressing cre recombinase and/or WT, R406H or G544D Munc18-1b with or without compound were subjected 7 days after lentiviral infection to an antibody uptake assay. Endocytosed synaptotagmin-1 antibody was quantified by immunostaining (A-C; scale bar = 30 µm; 4-PBA = 4-phenylbutyrate), via counting the number of pixels > intensity of 15 (D). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Dunnett's multiple comparison test; n = 3-10 independent experiments; exact n and p values are shown in Appendix Table S1).
Figure 3(A) Validation of PARN KO in HT1080 cells by Western blot. GAPDH is used as loading control. (B) In vitro deadenylation activity assay using protein extracts from HT1080 (WT) and HT1080PARN KO (PARN KO) cells. (C) TRAP assay using successive dilutions (500 ng, 250 ng, 125 ng and 62.5 ng) of cell extracts from WT or PARN KO cells. An internal control (IC) for PCR was used. (D) qRT-PCR analysis of hTR expression in WT and PARN KO cells. Expression levels were normalized first to ACTB and then to WT. Three independent RNA extractions were performed for each cell line. Error bars indicate s.e.m.. (E) Representative pictures of FISH against hTR in WT and PARN KO cells. Arrows indicate hTR foci. Scale bar 5 μm. (F) PARN detection by Western blot in the indicated conditions. Expression was induced by incubating cells with 10 ng/ml doxycycline for 72 h. Actin is used as loading control. PARN/actin ratios, normalized to doxycycline-treated WT/PARN cells, are shown below. (G) In vitro deadenylation activity assay using protein extracts from doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. (H) qRT-PCR analysis of the indicated gene transcripts in doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. Expression levels were normalized first to ACTB and then to WT/empty. Three independent doxycycline inductions were performed for each cell line. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied. (I) Representative western blot analysis of PARN, TRF2 and p53. Actin was used as loading control. Mean TRF2/Actin levels from two independent experiments are indicated below. (J) Same as (H) for p53 and p21 transcripts. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied. (K) qRT-PCR analysis of TERRA from the indicated chromosome ends in doxycycline-treated KO/empty and KO/PARN cells. Expression levels were normalized first to ACTB and then to KO/empty. Three independent doxycycline inductions were performed for each cell line. Error bars indicate s.e.m..
Figure 1A. Expression validation of mESC-specific SATS isoforms and their corresponding common isoforms by RT-PCR. The left panel shows the schematic structures of each isoform, with the first exons of the common isoforms and SATS isoforms named 1 and 1a (and 1b when multiple mESC-specific SATS isoforms were identified), respectively. Other exons are numbered according to their orders in the common isoforms. Due to space limitation, some canonical exons presented in both the common and SATS isoforms are omitted and represented by "//". The sites of forward primers (red) and reverse primers (blue) are marked at corresponding exons. mESC1 and mESC2 represent two different mESC lines; ADSC, adipose derived stem cells; NSC, neural stem cells; MEF, mouse embryonic fibroblasts; TTF, tail tip fibroblasts; NC represents PCR negative control without template. Numbers in parenthesis represent the abundance rank of SATS isoforms. B. Distribution of H3K4me3 ChIP-seq reads around the transcription start sites (TSS) of SATS (left) and common (right) isoforms in mESCs and MEFs. C. Distribution of Pol II ChIP-seq reads around the transcription start sites (TSS) of the SATS (left) and common (right) isoforms in mESCs and MEFs. D. Distribution of DNase-seq reads around the transcription start sites (TSS) of the mESC-specific SATS (left) and common (right) isoforms in three mESC lines (Ese14, Zhbtc4 and Escj7S) and three somatic cell lines (Nih3t3, Mel and fibroblast).
Figure 2NICD interaction with FBXW7 at endogenous levels in HEK293 cells. 500 µg of HEK293 cell lysates treated with DMSO or MLN4924 were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin served as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924. All data represent the mean ± SEM from three independent experiments. Student t-test was used to determine p values, with **p≤0.01.   Roscovitine treatment reduced the NICD-FBXW7 interaction. 500 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 together with Roscovitine were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin served as loading control.   Quantification of the density of western blot bands in (C) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924 treated samples. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with ***p≤0.001.
Mitochondrially anchored Vps41 can recruit AP-3.Figure 2(A) Model of Vps41 re-localization to mitochondria. In wild-type cells, AP-3 vesicles bud off the TGN and reach the vacuole where they are tethered by the HOPS subunit Vps41. Overexpressed Vps41, C-terminally tagged with GFP-Fis1, localizes to the mitochondria where it is able to tether AP-3 vesicles to the surface. A wild-type copy of Vps41 is still present in the background to minimize disturbance in the endogenous system. (B) Scheme of the Vps41 primary sequence with indicated motifs. The Vps41 protein harbours two motifs in its N-terminal region that have been shown to affect Apl5 binding, a PEST motif as well as an amphipathic lipid packaging sequence (ALPS) motif Furthermore, phosphorylation sites at residues S367, S368, S371 and S372 are shown, which are mutated in a phosphomimetic mutant (SD). (C) Tethering of AP-3 vesicles to the mitochondrial surface. Cells were grown at 30°C in YPG and kept in logarithmic phase. Just prior to imaging, the mitochondrial DNA was stained with DAPI Localization of Apl6 and Vps41 was analysed by fluorescence microscopy. First column shows DIC (difference interference contrast) image of cells. Scale bar, 5 µm. The inset shows the indicated magnification. Scale bar in inset, 2 µm. Apl5-dots co-localizing with GFP signal were counted and plotted in percent of the total number of Apl5-dots. Bars show the mean percentage of co-localization ± standard deviation (n ≥ 60 cells). (E, F) Purification of mitochondria from selected strains. Mitochondria were purified from the indicated strains as described in the methods. The amount corresponding to 1 % of the total volume was loaded of the 'load'' sample, 5% were used for 'crude' and 'pure' mitochondria fractions. Samples were loaded on a 10% SDS PAGE gel and analysed by Western Blot. White asterisks indicate unspecific bands. (G) Quantification of relative Nyv1 signal intensity from (F). Signal intensity was determined using ImageJ and a ratio of pure over crude signal intensity normalized to Tom40 signal intensity was plotted. Bars show the relative Nvy1 signal intensity ± standard deviation from n = 3 independent experiments.
AV formation after siRNA depletion of COPI requires Atg5 and Atg7.Figure 3.AV formation after siRNA depletion of COPI requires Atg5 and Atg7. 293/GFP-LC3 cells were incubated with siRNA for β′-, α-COP, or control siRNA alone or combined with siRNA against Atg5 or Atg7 for 48 h, then (A) lysed and analyzed by immunoblot for Atg5, Atg7, GFP-LC3, and tubulin (loading control), (B) fixed for immunofluorescence analysis using anti-ERGIC-53 antibodies, or GFP fluorescence. In A, the asterisk indicates nonspecific band; in B and C, asterisk indicates cells that did not show a fragmented ERGIC-53.
Figure 3(A) Immunofluorescence microscopy analysis of DRP1 localisation in control and subject fibroblasts. Mitochondria were visualized with TOMM20 (green) and endogenous DRP1 in red. Binary indicates thresholded binarized images and the hotmap shows thresholded and binarized images from DRP1 and mitochondria combined to create an image of mitochondrialDRP1. The mitochondrial DRP1 created image was then merged with the mitochondrial image. False colors show the accumulation of DRP1 at mitochondria. Scale bars: 10 µm. (B) Quantitation of the DRP1 localisation by immunofluorescence microscopy analysis. Upper panel: co-localization of TOMM20 and DRP1 in control and subject fibroblasts was calculated by Mander's and Pearson's coefficients. Bottom panel: quantification of DRP1 localisation in the cytosol and the mitochondria. At least 15 cells per condition were analyzed in 3 independent experiments. Errors bars represent mean +/- SD and p-values were calculated using a student t-test. (C). Analysis of DRP1 localisation in control and subject fibroblasts by immunoblot analysis after cellular fractionation. ITPR1 was used as an ER marker, VDAC1 as a mitochondrial marker and alpha-tubulin as a cytosolic marker. (D). Immunoblot analysis of mitochondrialDRP1 oligomers in control and subject fibroblasts, overexpressing an EGFP-tagged version of wild-type SLC25A46. Crude mitochondria were treated or not with the BMH cross-linker. SDHA was used as a loading control. Monomers (asterisk), dimers (arrowhead) and oligomers of DRP1 are indicated.
Toxoplasma-infected human macrophages die through AIM2-dependent atypical apoptosis (A) AnnV-Glo assay from IFNγ-primed THP-1 transfected with siRNA against indicated human NLR/ALR sensors and infected with type I or type II Toxoplasma gondii (Tg).Figure 3AnnV-Glo assay from IFNγ-primed THP-1 transfected with siRNA against indicated human NLR/ALR sensors and infected with type I or type II Toxoplasma gondii (Tg). Area under the curve (AUC) from real-time assays are plotted as mean ± SEM from n = 3 independent experiments. AnnV-Glo assay from IFNγ-primed primary human MDMs transfected with siRNA against AIM2 or non-targeting control (CTRL) and infected with type I or type II Tg. Area under the curve (AUC) from real-time assays are plotted from n = 4 independent experiments. Matched shapes and color of symbols indicate donors. Representative images (top) from IFNγ-primed THP-1 WT infected with type I Tg for 8 hours and stained for ASC and active caspase-8. Blue, Nuclei; Red, ASC; Green, active caspase-8 (IETD-FITC) and Grey, Tg. Scale bar, 5 μm or 2 μm in the magnified images. Quantification of ASC specks (bottom) in Tg-infected THP-1 WT or ∆CASP1 treated with IFNγ or left untreated at 8 hours post infection, plotted as mean ± SEM from n = 3 independent experiment. Immunoblots from primary MDMs infected with Tg strains for indicated times. Cells were untreated or pre-treated with IFNγ before infection. Images are representative of n = 4 biologically independent experiments.
MiR-16-5p mediates BDNF-induced dendritogenesis of hippocampal neurons.Figure 5Schematic of single-cell dual-fluorescence miRNA sensor assay. The control sensor plasmid consists of GFP and dsRed expressed under a separate promoter (top panel). The miR16 sensor includes two miR-16-5p binding sites at the 3'UTR of dsRed. Binding of endogenous miR-16-5p to the dsRed mRNA would inhibit the translation of dsRed but not GFP (bottom panel). B) Endogenous miR-16-5p activity in developing hippocampal neurons. Hippocampal neurons were transfected with 500ng miR16 or control sensor at 4 DIV and with or without LNA control ('LNA Ctrl') or the anti-sense miR-16-5p inhibitor ('LNA-miR16'). Cells were fixed at 7 DIV and the number of neurons expressing GFP and not dsRed ('GFP-only') was compared to the total number of transfected neurons. LNA-miR16 reduces the amount of repression of the miR16 sensor compared to LNA-Ctrl (n=4, p=0.02, t-test type 2, error bars represent s.d.). C) Short-term BDNF stimulation inhibits endogenous miR-16-5p activity. Hippocampal neurons were transfected at 4 DIV and treated with BDNF either 24h or 20 minutes before fixation and neurons were counted as in B. Repression index was obtained by normalizing the miR16 sensor to the control sensor condition in each experiment (n=4, p=0.02, t-test type 3, error bars; s.d.). D&E) Inhibition of miR-16-5p increases dendritic complexity and occludes BDNF-induced dendritogenesis. D) Hippocampal neurons were transfected with the control sensor (A), and stimulated with control or BDNF 24 hours before fixation. Pictures were obtained on a confocal microscope and thresholds were set to dsRed signal fluorescence in order to distinguish individual dendrites (scale bars are 10μm). E) To quantify dendritic complexity the total number of dendritic intersections was calculated using Sholl analysis. Values were normalized to LNA-Ctrl and control treatment (n=3, p<0.04, t-test type 3, error bars represent s.d.).F&G) Overexpression of miR-16-5p blocks BDNF-induced dendritogenesis. F) Hippocampal neurons were transfected with control sensor and either control miRNA mimic (miR-Ctrl) or a miR-16-5p mimic (miR16) and treated two days later with BDNF or control vehicle. DsRed signal fluorescence was used to set a threshold on images obtained on a confocal microscope (scale bars; 10μm). G) Total number of intersections was quantified in Sholl analysis and values were normalized to miR-Ctrl, control treatment in each experiment (n=4, p=0.02, t-test type 3, error bars; s.d.). H&I) Overexpression of the Dicer-binding deficient deletion mutant of TRBP (GFP-TRBP Δ) occludes BDNF-induced dendritogenesis. H) Hippocampal neurons were transfected with 200ng dsRed and either 50ng GFP or 200ng GFP-TRBP Δ, and treated two days later with BDNF or control vehicle. DsRed signal fluorescence was used to set a threshold on images obtained on a confocal microscope (scale bars; 10μm). I) Total number of intersections was quantified in Sholl analysis and values were normalized to control treatment in each experiment (n=5, p<0.02, t-test type 3, error bars; s.d.).
Figure 8 A. Confocal double immunofluorescence images depicting Olig2+ (a-e) and Olig2+-CC1+ (f-j) double labeled cells in the middle corpus callosum of control, cuprizone-treated mice at 5 weeks, in absence (cpz) or in presence of D-Asp (cpz + D-Asp), and 2.5 weeks after cuprizone withdrawal, in absence (vehicle) or in presence of D-Asp (D-Asp). (k, left panel), Quantitative analysis of Olig2+ cells (red) and Olig2+-CC1+ (blue) double labeled cells in the middle corpus callosum of control, cuprizone-treated mice, and cuprizone+D-Asp-treated mice at 5 weeks. (k, right panel), Quantitative analysis of Olig2+ cells (red) and Olig2+-CC1+ (blue) double labeled cells in the middle corpus callosum of control, vehicle, and D-Asp-treated mice 2.5 weeks after cuprizone withdrawal. Arrows point to Olig2+-CC1+ cells. Arrowheads point to Olig2+-CC1+ cells. Scale bar in a-j: 50 μm Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in A, k left panel, one-way ANOVA P<0.0001 followed by Newman-Keuls post hoc test, *P<0.05 versus ctrl; ^P<0.05 versus cpz (n=3 mice for each group). A, k right panel, one-way ANOVA P<0.0001 followed by Newman-Keuls post hoc test, *P<0.05 versus ctrl; ^P<0.05 versus vehicle (n=3 mice for each group  B. Representative electron microscopy images of middle corpus callosum sections from control mice (a), cuprizone treated-mice for 5 weeks in the absence (b) or in the presence of D-Asp (c), and from mice 2.5 weeks after cuprizone withdrawal in the absence (d, vehicle) or in presence of D-Asp (e). Scale bars in a-e: 1 μ  C. Percentage of myelinated axons in the middle corpus callosum of cuprizone (blue) and cuprizone + D-Asp-treated mice (red) for 5 weeks Data information: The values represent the means ± S.E.M. Level of significance was determined by using C, two-tailed Student's t test, *P<0.05 versus cpz (n=4 mice for each group  D. Scatter plot analysis of myelin thickness, expressed as individual G-ratios against axon diameters, after 2.5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle (blue circle) or D-Asp-treated mice (red triangle). Linear regression was used to indicate the differences between the groups in myelin thickness across the range of axon diameters  E. Histogram analysis displaying the G-ratio values of myelinated fibers across the range of axon diameters. Data represent the mean S.E.M. for at least 100 axons from each group Data information: The values represent the means ± S.E.M. Level of significance was determined by usin E, for each axon diameter group, Student's t test, *P<0.05 versus vehicle (n=4 mice for each group  F. Percentage of myelinated axons after 2.5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle (blue) or D-Asp- treated mice (red). Abbreviations: DEM, demyelination; REM, remyelination Data information: The values represent the means ± S.E.M. Level of significance was determined by usin F, for each axon diameter group, Student's t test, *P<0.05 versus vehicle (n=4 mice for each group)
Figure 6A LincRNA-EPS and negative control RNA (NC) were transcribed in vitro to label desthiobiotin and pulldown proteins from iBMMs lysate, the differential protein bands from silver-stained SDS-PAGE were cut for mass spectrometry analysis. One of the identified proteins from the band in the red box was PKR. B Western blot for verification of RNA pulldown-MS results with β-Actin as a negative loading control. C The interaction between PKR and lincRNA-EPS were checked by RIP-qPCR using anti-PKR antibody, and the efficiency of IP was verified by Western blot. D 20 nM siRNA targeting negative control (NC) and PKR were transfected into iBMMs for 48 h, the protein and mRNA expression level of PKR were tested by RT-qPCR and Western blot, respectively. E, F VSV titer in the WT and lincRNA-EPS-/- iBMMs transfected with siRNA as described in (D) and infected with VSV (MOI 0.1) for 8 h was measured by plaque assay (E). The alteration rates of viral titers were calculated in lincRNA-EPS-/- iBMMs relative to WT group (F). G VSV-N and VSV-G mRNA were transcribed from virus particles for RNA-pulldown, the interactions between VSV RNA with PKR protein was checked by Western blot, β-Actin was shown as a negative control. H LincRNA-EPS was transcribed in vitro and pulled down proteins from iBMMs infected with VSV for 4 h, PKR protein was detected by Western blot and GAPDH was shown as a loading control. I RNA-pulldown using biotinylated lincRNA-EPS from cell lysates of polyI:C (1 and 2.5 μg/mL) transfected iBMMs for 4 h, PKR protein was detected by Western blot and β-Actin was shown as a loading control. J HEK293T cells transfected with lincRNA-EPS and Flag-tagged PKR were infected with VSV to detect the PKR-binding lincRNA-EPS and VSV RNA by RIP-qPCR using anti-Flag antibody.