EMBO/sd-character-level-ner huggingface dataset:
epg-7 encodes a protein with similarity to mammalian FIP200 and yeast Atg11 and is degraded by autophagy.Figure 2.(A) Cloning of epg-7. (B) Protein sequence of EPG-7. The molecular lesions in identified mutant alleles are highlighted in red. (C) Schematic structures of EPG-7, FIP200, and Atg11. The coiled-coil domains are highlighted in green. The Atg11 domain is depicted in pink. (D) A large number of SQST-1::GFP aggregates accumulate in epg-7 mutant embryos carrying an epg-7(del Atg11) transgene. (E and F) No SQST-1 aggregates are detected in epg-7 mutants carrying an epg-7::gfp reporter. (E) DAPI image of the embryo shown in F. (G) EPG-7::GFP is weakly expressed and diffusely localized in the cytoplasm in wild-type embryos. (H-I) EPG-7::GFP is expressed in pharyngeal muscles (H, arrow) and cells in the tail region (I) at larval stages. (J) Compared with wild-type animals, levels of EPG-7::GFP are dramatically elevated in epg-4 mutants, but remain unchanged in sqst-1 mutants. (K) The expression level of EPG-7::GFP is dramatically elevated and many EPG-7::GFP aggregates are formed in lgg-1 mutant embryos. (L and M) No EPG-7 aggregates, detected by anti-EPG-7 antibody, are formed in wild-type embryos. (L) DAPI image of the embryo shown in M. (N) Levels of EPG-7 are dramatically elevated and a large number of EPG-7 aggregates are formed in atg-3 mutants. (O) No EPG-7 aggregates are formed in sqst-1 mutant embryos.
Figure 4A) Thyroid morphology was investigated using Nkx2-1 staining at E9.5 and E11.5 (sagittal sections) and at E13.5, E15.5, and E17.5 (transverse sections) in wild-type (wt) and Tubb1-/- littermates. Nkx2-1was used as a marker of early thyrocyte progenitors and differentiated thyrocytes. Delays in thyroid migration and in fusion of the median anlage to the ultimobranchial bodies were observed at E11.5 and E13.5 (arrow), respectively, in Tubb1-/- mice. At E11.5, stained migrating thyroid cells were visible along the tract only in Tubb1-/- littermates (arrow). At E17.5, a pyramidal lobe was visible in Tubb1-/- littermates (arrow). tr: trachea; ub: ultimobranchial body. Scale bar: 50 μ B) Total thyroid surface area (μm2) was quantified using Nkx2-1 staining at each embryonic stage. Top: thyroid surface area normalised for weight of each embryo. Bottom: thyroid surface area in μm2. Right: proliferation ratio calculated as the proportion of Nkx2-1-positive cells labelled with Ki67 at E9.5 in wt and Tubb1-/- thyroid anlages. ma: median anlage. Note that the thyroid ma is larger and the proliferation ratio higher at E.9.5 in Tubb1-/- mice, whereas from E13.5 onwards the Tubb1-/- thyroids were hypoplastic. Wt in black, KO, Tubb1-/- in gre C) Thyroid marker expression by quantitative PCR in thyroid tissue at E15.5 and E17.5 in Tubb1-/- versus wt mice normalised for peptidylprolyl isomerase A. Note the decreases in thyroid differentiation markers at E15.5 and E17.5 and also in thyroid transcription factors at E17.5 D) Staining of Nkx2-1 and T4 at E17.5 and surface area quantification demonstrating T4 retention in Tubb1-/- versus wt thyroids. The data are the percentage of T4 or calcitonin (CT) surface area versus total thyroid area (estimated from the Nkx2-1-stained surface area). Scale bar: 50 μm. Student"s test, *p<0.05, **p<0.01 and ***p<0.001. Experiments were done on at least three tissues per stage for each gen
Figure 4Scheme of the encapsulation setup and illustration, on the right, of the distribution of species within the encapsulated LLPS system. B-D. Representative confocal images of the microdroplets without (B) and with GTP (C and D). The concentrations of FtsZ, SlmA and SBS were 12, 5 and 1 μM, respectively (B and C) or 6, 3 and 0.5 μM respectively (D). 1 mM (C) or 2 mM GTP (D). Scale bars: 40 μm except in images on the far right (20 μm), which are either a magnification of the indicated region in the merged image (B) or independent images at higher magnification (C, D).
Role of yNap1 in nucleosome assembly on a genomic scale.Figure 1A Analysis of MNase-digested chromatin DNA fragments from wild-type (WT, sizes indicated in black) and nap1Δ (N, sizes indicated in red) cells by agarose gel electrophoresis. Diagram illustrating reduction of array length in nap1Δ cells. B Frequency distribution of nucleosomal DNA size (WT in grey fill with nap1Δ in blue traces) from H3 ChIP-seq. C Calculation of nucleosome fuzziness as the standard deviation of nucleosome (or tag) positions. See also Appendix Fig S1.
Figure 2(A) Overlay histograms of Jurkat-S staining with different human sera diluted 1:50. A pre-COVID19 serum sample is taken as negative control (grey histogram). (B) Bar plots of flow cytometry data generated with Jurkat-S cells and the panel of serum samples of Table EV1 classified according to their result in the indicated commercial tests: green, positive for the test; orange, weak or unclear; magenta, negative samples. Flow cytometry data is expressed as the ratio between the MFI of the antibody anti-S and the MFI for EGFR. Negative values for the flow cytometry test are those with a S/EGFR MFI ratio lower than 0.5. This ratio was set in order to fit most of the data negative for the other serological tests (pink triangles) under that threshold.Data represent the mean±s.e.m. All datapoints are shown.
Figure 1(A) Schematic representation of EB1 constructs and domain functions. CLIP170 (lower) uses CAP-Gly domains to interact with EB1's EEY motif, and secondary SxIP-mediated interactions with EB1's EBH domain. (B) Representative IF images in CHME3 stably expressing Flag alone control or the Flag-tagged EB1 constructs depicted in A co-stained for Tyrosinated-MTs (Tyr-MTs), and the nucleus (Hoechst). Scale bar, 10 μm. (C) HIV-1-VSV-luc infection is enhanced in CHME3 depleted of endogenous EB1 and stably expressing full-length (FL) EB1, but not in control Flag or any of the other EB1 constructs depicted in A. (D) Increasing amount of FL-EB1, but not ∆EEY results in a dose-dependent enhancement of HIV-1-VSV-luc infection in transiently transfected CHME3 cells. HIV-1-VSV-luc (E) infection is reduced in differentiated THP-1 depleted of CLIP170 determined by luciferase assays or FACS analysis, respectively. HIV-1-VSV-ZsGreen at various dilutions (corresponding to multiplicities of infection (m.o.i) 0.06, 0.03 and 0.02) (F) infection is reduced in differentiated THP-1 depleted of CLIP170 determined by luciferase assays or FACS analysis, respectively. (G) HIV-1-WT-luc infection is decreased in CLIP170 depleted CHME3 cells.
Figure 4(A) Comparison of the self-interaction of FAM134B WT with mutants as indicated using co-IP. SA-G216R was constructed by simultaneously mutating S149, S151 and S153 to alanine (SA) on the basis of FAM134B-G216R. (B) In vitro liposome fragmentation assay. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n≧3). (C, D) Measurement of the intracellular ER-scission activity of FAM134B WT, G216R, S151A-G216R and SA-G216R. GFP-tagged FAM134B proteins were expressed in U2OS cells at same levels. The autolyosomal degradation of ER fragments labelled by GFP-FAM134B were blocked by Baf A1 or DMSO. GFP-positive puncta were quantified for each cell in (D). For all of the groups, at least 30 cells were included for quantification; Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM. (E, F) Measurement of the ER-phagy activity of FAM134BG216R. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (G216R), mCherry-EGFP-FAM134B (S151A-G216R) or mCherry-EGFP-FAM134B (SA-G216R) at same levels. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (F). For WT, 50 cells were counted (n=50); for G216R, n=55; for S151A-G216R, n=51; for SA-G216R, n=52. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. *P<0.05, ***P<0.001, one-way ANOVA, error bars indicate SEM.
a) Fibroblasts of individuals with mucolipidosis II (I-cell disease; upper right panel) or Hurler syndrome (lower right panel) were compared with parental control cells (left panels).figf5(a) Fibroblasts of individuals with mucolipidosis II (I-cell disease; upper right panel) or Hurler syndrome (lower right panel) were compared with parental control cells (left panels). The alleles are indicated. Cells were fixed and stained with the indicated antibodies and DAPI to visualize DNA. Arrowheads point to the accumulated MRs. Scale bars are 20 μm. (b) Quantification of a (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).
Loss of sinhcaf increases acetyl-histone H3 (K9) and acetyl-histone H3 (K18) levels in the promoter region of kif26ab.Figure 6(A) Six pairs of walking primers were designed along the kif26ab promoter. (B) ChIP assay was performed to evaluate whether Sinhcaf binds to the kif26ab promoter directly in zebrafish. Data shown are mean ± SEM (n = 3, biological replicates), * p < 0.05. Statistical analysis was performed using 2-way ANOVA followed by Holm-Sidak post-hoc test. (C and D) The ChIP assay was performed to determine the influence of MZsinhcaf -/- mutation on acetyl-histone H3 (K9) and acetyl-histone H3 (K18) of the kif26ab promoter in zebrafish. Data shown are mean ± SEM (n = 3, biological replicates), * p < 0.05. Statistical analysis was performed using 2-way ANOVA followed by Holm-Sidak post-hoc test. (E) IGV track view of acetyl-histone H3 (K9) occupancy on the kif26ab gene region in MZsinhcaf mutant and wild type zebrafish embryos. The increased ChIP-seq peaks are highlighted with red dashed rectangle. (F) HEK293T cells stably expressing luciferase reporter construct driven by zebrafish kif26ab promoter were transfected with different amounts of CMV-Sinhcaf plasmid as indicated and luciferase activity was measured. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a,b) indicate a significant difference between the means, p<0.05.
Generation of hPI+MVF.Figure 5A Schematic illustration of human islet isolation and delivery. Islets are isolated from human donors and cultivated for up to 35 h until they are delivered for the generation of hPI+MVF. The entire process takes more than 8 days. B Bright field images of hFI, hCI, hPI and hPI+MVF. Scale bar: 100 µm. C Representative immunohistochemical stainings of insulin and CD31 in hFI, hCI, hPI and hPI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 100 µm. D-G Quantitative analysis of insulin-(D), glucagon-(E), somatostatin-(F) and CD31-positive cells (G) in hFI, hCI, hPI and hPI+MVF in % of all islet or islet organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. hFI; #P < 0.05 vs. hCI; +P < 0.05 vs. hPI. H Representative bright field images of angiogenic sprouting of hFI, hCI, hPI and hPI+MVF on day 3 of the sprouting assay. Scale bar: 150 µm. I Quantitative analysis of sprouting area of hFI, hCI, hPI and hPI+MVF in % of initial size (day 0) (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. hFI; #P < 0.05 vs. hCI; +P < 0.05 vs. hPI. J Quantitative analysis of C-peptide secretion (pg/mL) (normalized to DNA concentration) of hFI and hPI+MVF exposed to 1.1 mM and 16.5 mM glucose (n = 3 each). Mean ± SD.
Figure 4Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs. pre-MICU1: precursor form of MICU1; m-MICU1: mature form of MICU1 Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs. Where indicated, the cells were treated with 5 μM MG132 for 4 h. pre: precursor; int: intermediate; m: mature form Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with the indicated constructs, following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. The cells were treated with 5 μM MG132 for 4 h before fractionation Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with MICU1 WT-HA or MICU1 SD-HA, together with MCU-flag and EMRE constructs, following carbonate extraction at pH 7.4, pH 10.5, pH 11.5 or pH 12.5. The cells were treated with 5 μM MG132 for 4 h before fractionation ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs. Cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times, collected and subjected to western blotting analysis as indicated. In the graph, the amount of HA (MICU1), both WT and mutants, is represented relative to the amount at time 0. (n=3 independent experiments) ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs. Cells were harvested, and total protein was extracted and subjected to western blotting analysis with the indicated antibodies. Immunoblotting was performed under reducing (with the presence of DTT) or non-reducing (non-red) conditions (without DTT). Cells were treated with 5 μM MG132 for 4 h before lysis Quantification of HA levels in non-reducing condition showed in (E). (n=3 independent experiments) Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=3 independent experiments; 46-54 cells).
Figure 1The structure of a mitoTev-TALE. It consists in a mitochondrial localization sequence (MLS) and a TALE-DNA binding domain which is fused to a monomeric endonuclease (I-TevI), through a flexible linker (linker) Diagram of mtDNA illustrating the binding site for two monomeric mitoTev-TALEs (8.5 and 11.5 RVDs) designed to target the m.8344 A>G in the tRNALys gene. The two mitoTev-TALEs differ in the number of RVDs. The A:T base pair at position T0 is shown in green and the G:C discriminatory base pair in red. The I-TevI endonuclease domain targets a 5'CNNNG3' sequence, in this case 5'CACTG3' shown in green which is spaced by 12 bp from the TALE-DNA binding site HEK293T cells were transfected for 48 hours with each mitoTev-TALE (11.5 and 8.5 RVDs) and total cell extracts were used for western-blots with anti-T2A antibody. Molecular weights of the two different mitoTev-TALEs are ~85KD and ~70KDa, respectively Immunocytochemistry using the same antibody along with Mitotracker red was performed in COS7 cells, 24 hours after transfection. Nuclei were counterstained with DAPI (blue) 11.5RVD and 8.5RVD mitoTev-TALEs both co-localized with mitochondria, as seen in the merge image. Scale bar: 10 µm
Figure 2A) Representative confocal immunofluorescence and bright field (BF) images of HCT DKO cells expressing tBID-GFP (in green) treated or not with COMBO (ABT737 + S63586). Cyt c shown in magenta and nuclei in cyan. Scale bar 10 µm. B) Effect of tBID-GFP and mCherry-BCLXL on cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n> 30 cells per condition per experiment. *** p<0,001, ** p<0,025 and * p<0,05 with respect to tBID-GFP in HCT AKO condition. C) Representative confocal immunofluorescence images of HCT DKO cells upon indicated treatments. Nuclei (cyan) and cyt c (magenta) are shown. Scale bar 10µm. Arrowheads represent cells showing released cytochrome c and pyknotic nuclei. D) Representative WB showing the effect of TRAIL stimulation on endogenous BID cleavage in HCT DKO cells. n=3 independent experiments. E) % of cell death induced by TRAIL (1µg/ml), COMBO and their combination at different treatment durations on HCT DKO measured as in panel B. Paired Student's t-test ** p<0,025 and * p<0,05 with respect to MOCK condition. F) Effect of BID silencing on cell death upon TRAIL/COMBO treatments, measured as DRAQ7+ and normalized to siRNA control cell death. n=3 independent experiments. *** p<0,001 with respect to control condition. G-H) Representative WB of BID levels after silencing at different time points (G) or when genetically knocked out (H). G, n=3 independent experiments. I) Effect of TRAIL (1µg/ml), COMBO and their combination on HCT DKO and TKO cell death at different time points normalized to control cells. Each dot corresponds to the average of 3 technical replicates from 4 independent experiments. *** p<0,001. J) Representative HyVolution - superresolution images of TFAM-RFP expressing U2OS BAX/BAK DKO cells in the in the absence (top) and presence of tBID-GFP (bottom). TFAM appears in red, TOM20/mitochondria in grey and tBID-GFP in green. K) Quantification of cells with TFAM release on U2OS BAX/BAK DKO cells transfected with GFP-BAX/tBID-GFP and TFAM-RFP in the presence/ absence of COMBO. Dots correspond to individual experiments (n=3 individual experiments with more than 10 cells per condition per experiment). Unpaired Student's t-test *** p<0,001 with respect to non-treated conditions.
Figure 4(A) Representative images of mock- or ZIKV-infected human neurospheres expressing control (NTC) or FANCC-targeting siRNAs at 1-4 days post-infection. Scale bar = 250 µm. (B-C) Violin plot (B) and box plot (C) of relative size of neurospheres treated as described in (A) and measured on day 4 (B) or days 1-4 (C). Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. ***p < 0.001 by Student's t test. (D) Pie chart displaying the percentage of early apoptotic, late apoptotic, or necrotic hNSCs at 48 h after mock infection (blue), ZIKV infection (green), or siFANCC transfection (red). (E) Model of the effects of ZIKV infection on ER stress, autophagy induction, and FA gene expression leading to impaired virophagy and mitophagy.
Figure 4Outline of the spatial scheme utilized to simulate synapse cytoskeletal dynamics. Cartoon on the left shows the spatial patterning of the two major actin networks: the lamellar F-actin network in the peripheral zone that would continuously undergo fluctuations, and foci. The foci are interspersed throughout the synapse within a dynamic lamellar F-actin network. The peripheral lamellar fluctuations seen in live imaging could not be included in the simulations due to unavailability of data on fresh boundary adhesions. The cartoon on the right shows square simulation space patterned after the cartoon on the left and utilized here for simulations. The actin nucleation and adhesion parameters used at the foci and lamellar zones are indicated on the right. Simulation of T cell IS F-actin behavior and resultant mechanical tension incorporating the differential dynamics and positioning of foci and lamella across the synaptic interface, using Brownian dynamics equation with no inertia (see 'Methods'). The images show simulation snapshots at the beginning (0 min; soon after the attachment and spreading of T cells on the substrates) and the end of the simulations for the three IS cases - with persistent foci (WT, left most panels), with no foci (WASP-/-, middle right panels), and with WASP and consequently foci downregulated after a period of synapse maturation (WT transitional). The colors in the images indicate myosin II motor (pseudocolored teal), F-actin crosslinking proteins (pseudocolored yellow), and the F-actin filaments (pseudocolored blue for the low tension and the red for thigh tension actin filaments); middle left image panels show the distribution of the aforementioned proteins at individual foci site at a higher magnification. Predicted stress (tension normalized to the area) profiles within the cytoskeletal architecture as the synaptic contact progresses in time, corresponding to the three cases shown in (D). Live imaging of a T cell using SIM shows the inter-foci connections (arrows in the inset) as predicted in the simulations; and their loss concomitant with loss of actin foci, as the T cell polarizes to initiate motility (compare insets between 0 and 120 sec). '0 sec' refers to the beginning of the observation of the cell, after it has attached to the substrate, spread and maintained synapse for 12 min. Scale bar, 5µm in main images and 1.5μm in the magnified insets. Ultrastructural visualization of detergent extracted WT and WASP-/- T cell synaptic cytoskeleton using platinum replica electron microscopy (top images). Each image shows individual synapse, dark zone in the middle of the synapse represents cell nucleus. Scale bar, 2.5μm. The bottom left micrograph is the magnified inset from WT synapse and shows actin foci (identified as globular cluster of short filaments in the inset, marked with arrows)-dependent interconnected cytoskeletal architecture that is associated with synapse symmetry as predicted in the model, and is lacking in the WASP-/- cell synapse (bottom right micrograph- magnified inset from WASP-/- synapse). Scale bars, 1μm.This experiment was repeated at least twice with similar results.
Figure 7(A) Western blot analysis PABPDHFR cells depleted of LSM1, DCP2 or XRN1 by siRNA-mediated knockdown. siGFP represents a negative control. (B) Phase-contrast images of PABPDHFR cells previously transfected with siRNAs targeting GFP (control), LSM1, DCP2 or XRN1 and cultured in the presence or absence of TMP for 48 hours. Total numbers of viable cells, denoted in the upper right hand corner of images, were quantified with acridine orange and propidium iodide and calculated as a percentage of PABPDHFR cells grown in the presence of TMP, along with the SEM. Scale bar, 100 μm.
Figure 1(A) Schematic of low intensity exercise regimen. (B, C) Exercise capacity of MCK-KO and WT mice from the low intensity regiment was conducted. (n = 4 WT, n = 5 KO mice, mean ±SEM, * p < 0.05, two-tailed student's t-test). (D) Schematic of high intensity exercise regimen. (E, F) Exercise capacity of MCK-KO and WT mice from the high intensity regiment (n = 5 mice, means ± SEM, two-tailed student's t-test).