EMBO/sd-character-level-ner huggingface dataset:
Figure 9 Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies. (A) Absolute EPSC amplitudes during a 10 Hz train. Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies.(B) EPSC amplitudes during a 10 Hz train normalized to the first EPSC. Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies.(C) Top: example traces of the currents evoked by 40 Hz stimulation. Bottom right: first 5 pulses of the 40 Hz train. Far right: transferred charge during 40 Hz train. Insert shows total charge transferred during entire 40 Hz train (M18WT: 2.63 ± 0.38 nC; M18Y473A: 2.93 ± 0.54 nC, t test, p = 0.6630). Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies.(D) EPSC augmentation is calculated by dividing the EPSC amplitude of a single pulse after a stimulation train by the amplitude of the first EPSC within this train. Mean augmentation after 5 Hz train (M18WT: 0.54 ± 0.05, n=8; M18Y473A: 2.88 ± 0.47, n=10; Unpaired t-test with Welch correction, p = 0.0008), 10 Hz train (M18WT: 0.70 ± 0.10, n=8; M18Y473A: 5.39 ± 0.77, n=10; Unpaired t-test with Welch correction, p = 0.0002) and 40 Hz train (M18WT: 0.93 ± 0.11, n=8; M18Y473A: 8.77 ± 1.52, n=10; Unpaired t-test with Welch correction, p = 0.0006). Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies.(E) A single action potential or sucrose application (500 mM, 3.5 sec.) was given before (naive) or two seconds after HFS (100 pulses at 40 Hz). Average EPSC charge M18WT (Naive: 70.9 ± 19.5 pC, after HFS: 75.4 ± 17.4 pC, n=12, Paired t-test, p = 0.4049). Average EPSC charge M18Y473A (naive: 10.3 ± 4.5 pC, after HFS: 46.2 ± 14.5 pC, n=13, Wilcoxon matched-pairs signed-ranks test, p = 0.0005). Average RRP size M18WT (naive: 1.73 ± 0.69 nC, after HFS: 1.34 ± 0.54 nC, n=12, paired t-test, p = 0.0557) Average HFS size M18Y473A (naive: 0.268 ± 0.075 nC, after HFS: 0.439 ± 0.107 nC, n=13, paired t-test, p = 0.0100). Average Pves M18WT (EPSC charge / RRP charge * 100) (Naive: 7.07 ± 0.76 %, after HFS: 10.6 ± 1.6 %, n=12, paired t-test, p = 0.0201). Average Pves M18Y473A (naive: 1.98 ± 0.72 %, 12.2 ± 2.8 %, n=13, paired t-test, p = 0.0021).
Figure 5Gene expression profile of MDA231-LM2 cells treated with paclitaxel (PAX) for 48 hours. Highlighted are examples of JNK signature genes that are induced by PAX treatment. Venn diagram showing overlap between genes that were significantly (P < 0.05) induced more than 1.5-fold in response to paclitaxel treatment of MDA231-LM2 cells and genes that were significantly (P < 0.05) induced in MDA231-LM2 cells after formation of oncospheres. Significant fold changes were determined by two-sample empirical Bayer tests with Benjamini-Hochberg correction for multiple testing. PAX. GSEA normalized enrichment scores of four independent mammary stem cell signatures that arise in response to paclitaxel treatment of MDA231-LM2 cells. Immunofluorescence analysis of p-c-Jun+ cancer cells in lung metastases from mice injected intravenously with MDA231-LM2 cells and treated with PAX at week 3 post injection. Lungs were harvested 72h after PAX treatment. Cancer cells express GFP as a marker. DAPI was used for nuclear staining. Scale bar, 50 µm. Quantification of cancer cells expressing p-c-Jun in panel D. Values are mean from three mice ± SEM. Nine metastatic foci were analyzed per mouse. Western blot analyzing phosphorylated JNK (p-JNK), JNK, phosphorylated c-Jun (p-c-Jun) and c-Jun in MDA231-LM2 cells treated with PAX for 48 hours. Tubulin was used as a loading control. SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of PAX qPCR triplicates ± SD. SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of doxorubicin (DOXO). qPCR triplicates ± SD. SPP1 and TNC expression in SUM159-LM1 cancer cells treated with 4 μM 5-Fluorouracil or 4 μM methotrexate. Cells were treated for 48 hours. qPCR triplicates ± SD. SPP1 and TNC expression in SUM159-LM1 cells treated with paclitaxel and JNKi. qPCR triplicates ± SD. SPP1 and TNC repression by JNKi, P < 0.05 for both vehicle and PAX-treated cells.
Figure 4(a, b) MCF10.DCIS.com cells were transfected with FLAG-empty vector (EV), constitutively active RAB2A-Q65L, or dominant negative RAB2A-S20N mutant. Cells were immunostained with anti-FLAG (red) and RAB7 (a, green) or anti-EEA1 (b) antibodies. Merged images with DAPI (blue) are shown on the right. Enlarged endosomes in the boxed region are highly magnified in the insets. Diameters of RAB7 positive vesicles are also shown. n, number of cells scored for each cell population. The RGB profiler plugin from ImageJ was used to plot colocalization in the lines drawn in the inset images. Bars, 10 μm, (inset 2 μm). (c) MDA-MB-231 cells were transfected with EGFP-fused EV, RAB2A-Q65L, or RAB2A-S20N. Cells are immunostained with anti-RAB7 (blue) and anti-MT1-MMP (red) antibodies, and their merged images are shown. Merged images are shown on the right. Enlarged endosomes containing MT1-MMP in the boxed region are highly magnified in the insets. Note that RAB2A-Q65L and RAB7-positive vesicles contain MT1-MMP (arrows). Bars, 10 μm, (inset 2 μm). (d-f) MDA-MB-231 cells expressing MT1-MMP-pHluorin transfected with scrambled siRNA (siNC) or siRNA against RAB2A were seeded on type I collagen, and images were acquired for 20 min period by TIRF microscopy. Frequency of MT1-MMP-pHluorin exocytic events was quantified (e). The corresponding time-lapse series are shown in Movie 1, while an entire cell shown in Movie 2. Efficacy of RAB2A gene silencing was verified by qPCR (f). Bars, 2 μm. **, P < 0.01.
Figure 3A. CRISPR-Cas9 strategy to derive HUVECs with an excision of the LE in the RAB13 3'UTR (∆LE) and parallel generation of wild type (Wt) control cells. The Wt RAB13 exon 8 is represented with its coding sequence in dark and the 3'UTR in clear boxes. The 5' and 3' gRNA-targeted regions are represented with green lines. Arrows: relative positions of the forward (f) and reverse (r) PCR primers used to identify HUVECs with CRISPR-Cas9-mediated excision of the LE. B. Representative genotyping PCR demonstrates the band size shift in ∆LE HUVECs. C. Detailed DNA sequence depicting nucleotide positions within the RAB13 3'UTR of Wt and ∆LE HUVECs. D. Wt and ∆LE HUVEC RNAseq mapped reads depicting RAB13 exon usage. E. Quantification of RAB13 mRNA smFISH spot number in Wt and ∆LE HUVECs (n=3 experiments; ns: not significant; unpaired t test). F. Number of RAB13 mRNA smFISH spots plotted against the respective Polarisation Index (PI) (n=29 cells; ns: not significant; linear regression). G. Left: representative Western blotting (WB) of Wt and ∆LE HUVECs. Right: densitometry analysis of WB data (n=3 samples; ns: not significant; unpaired t test). H. smFISH co-detection of RAB13 and control GAPDH in Wt and ∆LE motile HUVECs cultured under subconfluent conditions. Arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µm (H). Bar charts are presented as means±s.d. I. PI of RAB13 and GAPDH co-detected in Wt and ∆LE HUVECs (n=29 cells; P***<0.001, ns: not significant; Mann-Whitney test). J. RAB13 PI plotted against respective GAPDH PI. The slope of the coloured lines represents the average RAB13/GAPDH PI ratio; the dashed grey line represents a 1:1 ratio (n=29 cells).
Figure 3(a) Reaction scheme for complete firing factor recruitment reaction conducted on ARS305 linear DNA. (b) Recruitment reaction performed as described in (a). (c) Sld3/7 recruitment reaction conducted as in Fig. 2a. (d) Replication reaction performed as in Fig. 1g. (e) Representative tetrads from dissection of SLD3+/sld3-6E heterozygotes.
a,c) MEFs (a) or HeLa cells (c) stably expressing FLAG-Parkin and either EGFP-VAP-A or HA-VAP-A were treated with CCCP (30 and 10 μM, respectively) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to HA or to FLAG (M2).f2(a,c) MEFs (a) stably expressing FLAG-Parkin and either EGFP-VAP-A or HA-VAP-A were treated with CCCP (30 and 10 μM, respectively) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to HA or to FLAG (M2). The fluorescence of EGFP-VAP-A was also monitored directly. (b,d) Higher-magnification views of the boxed areas in a and c, respectively. (a,c) HeLa cells (c) stably expressing FLAG-Parkin and either EGFP-VAP-A or HA-VAP-A were treated with CCCP (30 and 10 μM, respectively) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to HA or to FLAG (M2). The fluorescence of EGFP-VAP-A was also monitored directly. (b,d) Higher-magnification views of the boxed areas in a and c, respectively. (e) NIH 3T3 cells stably expressing EGFP-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, after which whole-cell homogenates as well as cytosolic, ER and mitochondrial fractions were prepared and subjected to immunoblot analysis. Scale bar (a-d), 10 μm.
Drug combinations synergistically induce the integrated stress response A Relative ATF4 and CHOP mRNA expression levels in HCT116 cells upon the indicated treatments determined by qPCR (NEN (N) 1.Figure 2A Relative ATF4 and CHOP mRNA expression levels in HCT116 cells upon the indicated treatments determined by qPCR (NEN (N) 1.2 µM; Domperidone (Domp, D), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM). Data information: Data are presented as mean (SD) and were analyzed by one-way ANOVA with Tukey post-hoc test (A, upper left and lower left graphs, (N=3), (D (N=3)), Kruskal-Wallis with Dunn´s post-hoc tests (A, upper right and lower right graphs, (N=5, besides NEN+Clomi (N=4) and NEN+Desi (N=4)) In (A), right graphs, significance is indicated for the comparison of combinatorial treatments (NEN+TCAs) to controls. * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001. B Immunoblot analysis of CHOP, ATF4, phosphorylated (p-) and t-) eIF2alpha (eIF2a) of HCT116 cells treated as indicated for 16h (NEN (N) 1.2 µM; Domperidone (Domp), Amitriptyline (Ami), each 30 µM). C Caspase activity in HCT116 cells either transfected with control (Ctrl), ATF4- or CHOP-targeting siRNAs and treated as indicated (NEN (N) 1.2 µM, Domperidone (D) 30 µM). Data information: Data are presented as mean (SD) and were analyzed by two-way ANOVA with Tukey post-hoc test (C (N=3), E (N=4), F (N=4). D Relative mRNA expression levels of the indicated genes upon drug treatments (Control (Ctrl), NEN (N) 1.2 µM, Domperidone (D) 30 µM) and inhibition of the ISR using ISRIB (1 µM), determined by qPCR. Data information: Data are presented as mean (SD) and were analyzed by one-way ANOVA with Tukey post-hoc test , (D (N=3)), E Caspase activity in HCT116 cells grown as monolayer (2D) and treated as indicated for 24h (Control (Ctrl), NEN 1.2 µM, Domperidone (D) 30 µM, ISRIB 1 µM). F Ratio of toxicity and viability in HCT116 spheroids treated as indicated for 48h (NEN 1.2 µM, Domperidone (Domp), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM, ISRIB 1 µM).
a) Depiction of FPP assay.figf5(a) Depiction of FPP assay. Digitonin selectively permeabilizes the plasma membrane but not internal membranes. Proteinase K degrades cytoplasmic components but membranous compartments remain intact. Under these conditions, MKLP1-GFP-labelled MBds (blue ellipse) in the cytoplasm will be degraded whereas those inside membrane-bound compartments will not. (b) MBds in MBd-poor hRPE-1 cells are largely protected (∼90% in membranous compartments, 10 cells analysed), whereas most MBds in HeLa cells are not (∼27%, 11 cells analysed), and are thus degraded in cytoplasm. Scale bar, 5 μm. (c) Presence of MBds in lysosomes on chloroquine or E64d-pepstatin A (E64d-PepA) inhibition in hRPE-1 and HeLa cells, but not in MCF-7 and H9 hESCs. Chloroquine treatment of H9 hESCs is not included as it caused differentiation and cell death. A representative image of hRPE-1 cells inhibited by chloroquine is shown, depicting two MBds inside lysosomes. MKLP1 and LAMP2 are used as MBd (red) and lysosome (green) markers, respectively. DAPI, DNA (blue). n=100 MBds per treatment in each of the biological triplicates. Scale bar, 5 μm. (d) Percentage of MBd+ cells (MBd levels), percentage of MBds within lysosomes and percentage of cells exiting cytokinesis following synchronization. MKLP1 and LAMP2 are used as markers as in c. Note that MBds are transferred into only one of the two nascent daughter cells after abscission (Fig. 2d), so a 50% maximum will be expected for MBd+ cells. The peak of MBds transferred to cells is 3 h after plating followed by a peak of MBds entering lysosomes at 7 h. (e) Both chloroquine and E64d-PepA treatments increase the percentage of MBd+ cells in hRPE-1 cells and HeLa cells (chloroquine, P=0.0021 and P=0.0187, respectively; E64d-PepA, P=0.0022 and P=0.0043, respectively; n=3 for all experiments). In contrast, lysosomal inhibition has no detectable effect on hESCs (H1, H9) or MCF-7 cancer cells. DMSO, dimethylsulphoxide. Data are presented as mean±s.d. (c-e), except mean±s.e.m. in hESCs (e).
Comparison of FRH and Mtr4 structures.Figure 2(A) FRH large cell (cyan), Mtr4 (pdb 2XGJ, green, RNA backbone in orange), and FRH small alternate cell (blue) differ primarily in the orientation of the arch domain. The FRH RNA-bound structure also crystallizes in the small-cell configuration. The two different FRH structures have an overall RMSD of 3.9 Å and the RMSD between Mtr4 and FRH is 2.9 Å (over 734 residues, for large cell FRH). (B) SAXS data indicates that FRH-Δ114 is monomeric and flexible. The crystal structure of the large cell structure predicts the solution scattering curve (left, χ2 = 0.92) and fits well to the calculated molecular envelope (right).
Figure 4a: Dose response curve for the ISA D614 and for the mCherry D614 strains obtained by fluorescence or viral RNA measurement in VeroE6 cells from one representative experiment. Data are represented as mean ± SD (indicated by the error bars). Each experiment was performed in technical triplicates (N=3). b: Table of EC50 values obtained for the two different strains from two technical replicates. and their respective mean ± SD. c c: Fluorescence of the SARS-CoV-2 mCherry in VeroE6 cells with different Remdesivir concentration. Scale bar, 200µM.
a, Immunohistochemistry of DRG neurons from Atg5+/+ and Atg5-/- mice at different developmental stages (embryonic day (E)13.figf4a, Immunohistochemistry of DRG neurons from Atg5+/+ and Atg5-/- mice at different developmental stages (embryonic day (E) 13.5, E15.5 or postnatal day (P) 0), stained with an antibody directed against ubiquitin. Scale bar, 25 µm. b, Accumulation of Triton-X-100-solublepolyubiquitinated proteins in the brains of Atg5flox/flox; nestin-Cre mice. Brain homogenate prepared at the indicated times from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice were separated into Triton-X-100-soluble (S) and -insoluble (P) fractions and analysed by immunoblotting using anti-ubiquitin antibodies. Arrowhead indicates the stacking gel. c, Immunohistochemistry of liver sections from control (Atg5flox/+; CAG-Cre) and Atg5flox/flox; CAG-Cre (Atg5flox/flox) mice at four months of age, using an anti-ubiquitin antibody. Scale bar, 25 µm. d, Immunohistochemistry of liver sections from six-week-old Atg5flox/flox; Mx1-Cre mice at the indicated time points after pIpC injection, using anti-ubiquitin antibodies. Arrowheads indicate ubiquitin-positive inclusion bodies. Scale bar, 25 µm. e, Five thousand hepatocytes were randomly selected, and the number of cells with diffuse cytosolic ubiquitin signals, with (black) or without (white) inclusion bodies, were counted.
Silencing dGcn5 facilitates the clearance of Tau aggregates A, B The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding.Figure 7The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding. The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR-Gal4 driver. Drosophila (GMR-Gal4/+) was used as the control. The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding. The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR-Gal4 driver. Drosophila (GMR-Gal4/+) was used as the control. C Representative light micrographs of adult Drosophila eyes expressing the indicated transgenes under the control of the GMR-Gal4 driver. The Drosophila were fed with or without CQ. Drosophila (GMR-Gal4/+) was used as the control. D Distribution of the different eye phenotypes following genetic manipulations The Drosophila were fed with or without CQ (30 flies per condition were randomly selected for analyses; *p < 0.05, ***p < 0.001, ꭓ2 test).
Figure 3A. FLAG-DnaJC5 overexpression facilitates extracellular release of wildtype (WT), P301L and R406W tau by dot blot in HEK293T cells. Intracellular protein levels are shown by Western blot; DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels shown below; data are mean ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. B. Overexpression of FLAG-DnaJC5 induces extracellular release of wildtype (WT) and A53T α-synuclein in HEK293T cells. Extracellular protein levels shown by dot blot (quantification below, mean ±SEM, n=3 ***p<0.001, ** p<0.01 by one-way analysis of variance with Tukey's multiple comparison post-hoc analysis) and intracellular proteins levels were analyzed by Western blot, DnaJC5 was detected by FLAG antibody. C. DnaJC5 overexpression induces extracellular release of GFP-tagged wildtype (WT), A315T and Q343R TDP-43 in HEK293T cells. Intracellular DnaJC5 was detected by FLAG antibody. Extracellular protein levels are analyzed by dot blot (quantification below, mean ±SEM, n=4) ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.
ATP destabilises the pre-AMPylation complex and the FICD dimer.Figure 6A BioLayer interferometry (BLI) derived association and dissociation traces of monomeric FICDL258D-H363A (mFICDH363A) or dimeric FICDH363A (dFICDH363A) from immobilised biotinylated BiPT229A-V461F in absence or presence of nucleotides. Unless indicated (*) BiP was saturated with ATP before exposure to FICD variants. A representative experiment of three independent repetitions is shown. B BLI dissociation traces of proteins as in (A). At t = 0 a pre-assembled complex of immobilised, ATP-saturated BiP and the indicated FICD proteins (associated without ATP) were transferred into a solution without or with ATP, as indicated. A representative experiment is shown and the biphasic dissociation kinetics are quantified in (C) and (D). C Graph of the slow dissociation rates (koff,slow) of monomeric FICD from BiP:ATP as shown in (B). Bars represent mean values ± SD of three independent experiments. D The ATP-induced fold change in the percentage of the dissociation phase attributed to a fast dissociation (%Fast), koff,fast, and koff,slow derived from the data represented in (B). Bars represent mean values ± SD of three independent experiments. E (i) BLI assay workflow used to derive FICD dimer off rate data presented in (ii) (ii) Observed dimer off rates under different nucleotide conditions (5 mM, where applicable). ATP, but no ADP, significantly increases the dimer dissociation rate [n.s.: not significant by Tukey test]. Data shown is the mean ± SD of four independent experiments. F Validation of the homo-FRET dimerisation probe. The dimerisation sensitive TMR fluorescence of the labelled FRET dimer (2.5 nM) is de-quenched specifically by equilibration with excess unlabelled dimerisation-competent FICDH363A but not monomeric FICDL258D-H363A. Mean ± SD of three independent experiments. G Homo-FRET measurement of nucleotide dependent modulation of the FICD monomer-dimer equilibrium. ATP increases and ADP decreases the proportion of monomeric FICD. * Plateaus were constrained to a shared best fit value. Data shown is the mean ± SD of four independent experiments.
Figure 7A. Cartoon of the contribution of TF in cytoplasmic protein folding (left) and secretory protein targeting (right, black arrows). The secretory targeting factors SecB and SecA that act independently (grey arrows) or interface with TF are also shown. Blue,cyan: other folding chaperones. B. Schematic model of TF:proOmpA targeted to the translocase with or without contributions from SecB and subsequent SecA C-tail/SecB-mediated control of proOmpA translocation. Oval to rectangle transition in SecA signifies conformational changes. B: SecB; cyto: cytoplasm; IM: inner membrane; peri: periplasm.
Figure 6(A) Venn diagram showing the total and overlapping numbers of genera enriched in the fer-4 mutant and under Pi starvation. Data information: All experiments were repeated three times with similar results. The fer-4 rhizosphere microbiome promotes plant growth and alleviates LP stress. Arabidopsis seeds were germinated on 1/2-strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days. The seedlings were then transferred to vermiculite and inoculated with Col-0 or fer-4 rhizosphere microbiomes. (B), Images were taken 4 weeks after inoculation. Data information: All experiments were repeated three times with similar results. The fer-4 rhizosphere microbiome promotes plant growth and alleviates LP stress. Arabidopsis seeds were germinated on 1/2-strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days. The seedlings were then transferred to vermiculite and inoculated with Col-0 or fer-4 rhizosphere microbiomes. (C), The seedlings were harvested, and the shoot fresh weight was measured 4 weeks after inoculation. The data shown indicate the means ± SDs (n = 7, n refers to the number of flowerpots (4 seedlings per flowerpot) per group). The different lowercase letters indicate statistical significance (one-way ANOVA). Data information: All experiments were repeated three times with similar results. (D) The fer-4 rhizosphere microbiome upregulated phosphate absorption gene expression. The roots of Col-0 were harvested after 4 weeks of inoculation with the Col-0 or fer-4 rhizosphere microbiome under LP condition, and the relative expression of Pi-absorbing genes was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; *, p < 0.05; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.