EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Fluorescence microscopy of human fibroblast cell lines derived from ZSD patients-cWT, cWT+ATAD1, PEX3-/-, and PEX3-/-+ATAD1-expressing PEX13-GFP and stained with Mitotracker Deep Red FM. Representative images are shown, scale bar, 10 μm. (B) Electron microscopy of human fibroblast cell lines cWT, cWT+ATAD1, PEX3-/-, and PEX3-/-+ATAD1. Representative images are shown, scale bar 500 nm. (C) Quantification of electron microscopy imaging. Mitochondrial length and width were analyzed using the Fiji length analysis tool. Per cell line >50 mitochondria were graphed. The horizontal lines represent the mean length and width in each cell line, respectively. (D) Quantification of electron microscopy imaging. Three independent experiments were quantified. Mitochondria were counted (cWT n = 232, PEX3-/- n = 266, and PEX3-/-+ATAD1 n = 257) and binned into 3 categories: mitochondria with many cristae and low electron density (Type 1), mitochondria with fewer cristae (Type 2), and mitochondria with high electron density and/or almost no cristae (Type 3).
Figure 5(A) Experimental design for artificial flickering. WT MEFs expressing Su9-GFP were subjected to repetitive FCCP treatments and observed using confocal microscopy in the presence of TMRE. (B) Three frames from the time-lapse analysis during artificial flickering are shown. The boxed regions are magnified. Scale bar, 5 µm. (C) The fluorescence intensity of Su9-GFP and TMRE in mitochondria was quantified. (D) Western blotting of WT MEFs after the indicated FCCP treatments. The asterisk indicates non-specific bands of anti-Oma1 antibodies.
Figure 7 (A) Life span analysis of flies of the indicated genetic background. At least 50 flies were analysed for each group. Log-rank test (Mantel-Cox test, Mantel-Haenszel test, p < 0.0001)  (B) Graph bar shows mean± SEM of the climbing performance of flies of the indicated genotypes. One way ANOVA, Newman Keuls test, n=3  (C) Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes. Graph shows (mean± SEM, n=3 independent experiments) respiratory control rate (RCR) calculated as described. One way ANOVA, Newman Keuls test  (D) Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes. The experiment was repeated three times in replicates. Bar graphs represent quantification of electron dense mitochondria (represented as grey bar) and mitochondrial with ruptured cristae (represented as white bars) from the indicated genotypes
Application of yeast-based TAR cloning to generate viral cDNA clones and the recovery of recombinant MHV-GFP.Figure 1.Application of yeast-based TAR cloning to generate viral cDNA clones and the recovery of recombinant MHV-GFP.a, Schematic representation of the general workflow of TAR cloning and virus rescue. In-yeast assembly require the delivery of overlapping DNA fragments covering the viral genome and a TAR vector into yeast in one transformation event. Transformed DNA fragments are joined by homologous recombination in yeast generating a YAC-cloned full-length viral genomic cDNA. The in vitro production of infectious capped viral RNA starts with the isolation of the YAC, followed by plasmid linearization to provide DNA template for run-off T7 polymerase-based transcription. Virus rescue starts with electroporation of BHK-21 and/or BHK-21 cells expressing the corresponding coronavirus N protein, followed by co-cultivation of electroporated cells with susceptible cell line for virus production and amplification. Schematic representation of MHV-GFP genome organisation with 9 viral subgenomic overlapping cDNA fragments used for TAR cloning. pT7, T7 RNA polymerase promoter; UTR, untranslated region; An, poly (A) tail; J represents assembly junction between two overlapping DNA fragments. Recovery of infectious recombinant MHV-GFP. Cell culture supernatants containing viruses produced after virus rescue from two MHV-GFP YAC clones (Clone1, Clone 2) were used to infect 17Cl-1 cells. At 48 hours post-infection, infected cells were visualised for GFP expression (left panels) and by bright field microscopy (right panels). Mock represents 17Cl-1 cells inoculated with supernatant from BHK-MHV-N cells electroporated without viral RNAs. Replication kinetics of parental MHV-GFP and recombinant MHV-GFP Clone 1 and Clone 2. L929 cells were infected at an MOI of 0.1. Cell culture supernatants were harvested at indicated time points post-infection and titrated by plaque assay. Data are representative of three independent experiments with two replicates per virus in each experiment. Error bars show standard deviation (SD) from the mean. pfu/ml, plaque forming units per millilitre.
Shown are the numbers of individual siRNAs from a pool of four targeting each gene that scored positive in each screen.f2Shown are the numbers of individual siRNAs from a pool of four targeting each gene that scored positive in each screen. Red, genes with 2 or more positive siRNAs (confirmed hits); Green, genes with 2 positive siRNAs. See Supplementary Tables 2-8 for further details.
Isolation and identification of the SARS-CoV-2 virus.Figure 1.(A) Vero cell was inoculated with BALF sample, and the CPEs were observed at 4-dpi. (B) RNAs were extracted from the cell culture supernatant, and detected using a commercial kit targeting the ORF 1ab (red) and N (blue) genes of SARS-CoV-2. Testing the convalescent plasma IgG antibody (C) using the purified SARS-CoV-2 particles. The control plasmas 1 and 2 were obtained from a patient recovered from influenza A virus infection and a healthy volunteer, respectively. The control monoclonal antibody is a human monoclonal antibody specific to influenza A virus generated by our laboratory. The PBS control was serum-free. Testing the SARA-CoV-2-RBD specific human monoclonal antibodies (D) using the purified SARS-CoV-2 particles. The control plasmas 1 and 2 were obtained from a patient recovered from influenza A virus infection and a healthy volunteer, respectively. The control monoclonal antibody is a human monoclonal antibody specific to influenza A virus generated by our laboratory. The PBS control was serum-free. (E) Viruses were detected by IFA using the patient's plasma, and plasma from a healthy control was used as negative control. Scale bar, 100 μm. (F) Phylogenetic characteristics of BetaCoV/Shenzhen/SZTH-003/2020, and this isolate was indicated with a triangle.
Autophagic response in cells with compromised respiratory chain complex III, IV, or V activity during amino‐acid starvation.Figure 2Autophagic response in cells with compromised respiratory chain complex III, IV, or V activity during amino‐acid starvation. Wild‐type, rho0, atg7 cells and mutants that are selectively inhibited in the biogenesis of respiratory chain complex III (CIII: cbs1), complex IV (CIV: mss51, pet111, pet122), or the F1Fo‐ATP synthase (complex V; CV: atp10) were exposed to amino‐acidstarvation medium supplemented with acetate (A) or galactose (B). When indicated, wild‐type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino‐acid starvation period. All strains expressed prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels). Samples were analysed as described in Figure 1A. The means and s.d. of five (n=5) independent experiments are indicated.
Figure 3Scheme representing the two cell lines generated from the mutant TDP-43 iPSCs by either endogenously tagging the WT TDP-43 allele or the mutant TDP-43 allele with mCherry. Immunofluorescent analysis with in C its corresponding intensity plot showing cytoplasmic mislocalization of the MUT-mCherry TDP-43 in motor neurons, but not in WT-mCherry TDP-43. Scale bar: 20µm. Quantification of nucleo-cytoplasmic (N/C) intensity ratio based on immunohistochemistry of WT-mCherry (n=45) and MUT-mCherry (n=63) tagged TDP-43. Each dot represents one analysed cell, unpaired Mann-Whitney-test. Western blots showing nucleo-cytoplasmic fractionation of WT-mCherry and MUT-mCherry iPSC-derived motor neurons with F (left panel) quantification of total mCherry and total untagged TDP-43 on α-tubulin and (right panel) relative cytoplasmic mCherry and untagged TDP-43 on total mCherry and total untagged TDP-43 respectively quantification of total mCherry and total untagged TDP-43 on α-tubulin and (right panel) relative cytoplasmic mCherry and untagged TDP-43 on total mCherry and total untagged TDP-43 respectively, ratio paired-t-test. (left panel) quantification of total mCherry and total untagged TDP-43 on Histone H4 and (right panel) relative nuclear mCherry and untagged TDP-43 on total mCherry and total untagged TDP-43 respectively, ratio paired-t-test. Quantification of nucleo-cytoplasmic (N/C) intensity ratio based on immunohistochemistry of WT-mCherry (n=45) and MUT-mCherry (n=63) tagged TDP-43. Each dot represents one analysed cell, unpaired Mann-Whitney-test. Western blot showing untagged TDP-43 (left panel) and phosphorylation state of the untagged allele (right panel) in WT -and MUT-mCherry motor neurons and in I ratio quantification of normalized pTDP43/normalized TDP-43 (because of almost the same molecular weight two different blots were used), ratio paired-t-test.
Figure 6HeLaS3 and AGO2KO cells were transfected with a synthetic terc-sRNA or an siRNA with no target in humans (siCtl), as a control. Four days after transfection, telomerase activity in 100- and 50 cell-lysate was assessed by TRAP. Intensity of the telomerase products (6 bp-ladder) was determined by ImageLab Software (Biorad) and normalized with the intensity of the Internal Control (IC), (n=4 experimental replicates)
FMN2 is highly expressed in mossy fibers and is required for synaptic depotentiation.Figure 2Figure 2: FMN2 is highly expressed in mossy fibers and is required for synaptic depotentiation.A. Fmn2 expression was analyzed via qPCR in brain regions from 3-month old mice (Left panel, n=5) and in mouse primary hippocampal neurons, astrocytes (n=5) and human postmortem hippocampus (right panel; n=3/group). Figure 2: FMN2 is highly expressed in mossy fibers and is required for synaptic depotentiation.B. Representative immunoblot images showing FMN2 protein levels in the mouse hippocampus (Hip M), cerebellum (Cere M) and human hippocampus (Hip Hu) Figure 2: FMN2 is highly expressed in mossy fibers and is required for synaptic depotentiation.C. Representative immunoblot images showing FMN2 levels in the hippocampus of wild type and Fmn2 -/- mice Figure 2: FMN2 is highly expressed in mossy fibers and is required for synaptic depotentiation. D. Representative low (upper panel, scale bar 500 µm) and high magnification images (lower panel) showing FMN2 expression (green) in FMN-EGFP knock in mice.For all experiments parasagittal brain slices from 14-21 day old mice were used. Cere, cerebellum; Hip, hippocampus, mf; mossy fiber, str. Luc.; stratum lucidum, cc, corpus callosum. Figure 2: FMN2 is highly expressed in mossy fibers and is required for synaptic depotentiation.E. Left panel: Representative images showing the co-localization of FMN2 (green) and Synaptoporin in the mossy fiber pathway in low and high magnification. Right panel: Immunoblot analysis of hippocampal synaptosomes (Syn) compared to cell homogenate (H) showing that FMN2 is present in the pre-synaptic compartment. Synaptophysin was used to confirm enrichment of synaptosomes. For all experiments parasagittal brain slices from 14-21 day old mice were used. Cere, cerebellum; Hip, hippocampus, mf; mossy fiber, str. Luc.; stratum lucidum, cc, corpus callosum. Figure 2: FMN2 is highly expressed in mossy fibers and is required for synaptic depotentiation.F. LTP was similar in Fmn2-/- (n=12) and wild type control mice (n=8).Error bars indicate S.E.M. Figure 2: FMN2 is highly expressed in mossy fibers and is required for synaptic depotentiation.G. The absolute level of LTD was similar amongst Fmn2-/- and wild type mice (n=10). Error bars indicate S.E.M. Figure 2: FMN2 is highly expressed in mossy fibers and is required for synaptic depotentiation.H. Depotentiation was significantly impaired in Fmn2-/- mice (n=12) when compared to control (n=11; *P < 0,05). Error bars indicate S.E.M.
Figure 1(A) Topo II activity on random juxtapositions of DNA segments (*) produces steady-state fractions of DNA knots in intracellular chromatin. (B) DNA knotting probability (Pkn) of intracellular chromatin (observed) does not scale proportionally to the length of DNA (expected). The slope of Pkn is reduced in chromatin stretches larger than 20 nucleosomes. Data from (Valdes et al., 2018). (C) Three-gate mechanism of topo II to pass one segment of DNA (T-segment) through another (G-segment). Upon ATP binding, the T-segment is captured by the entrance gate (N-gate) and passed through the transiently cleaved G-segment (DNA-gate). Upon re-ligation of the G-segment, the T-segment is released through the exit gate (C-gate). (D) Topo II activity reduces the fractions of DNA supercoils, knots and catenates to below the topological equilibrium values (see details in Fig EV1). (E) Architecture of the SMC complexes of S. cerevisiae. The Smc heterodimers (Smc1-Smc3, Smc2-Smc4, Smc5-Smc6) and kleisin (Scc1, Brn1, Nse4) subunits of cohesin, condensin and the Smc5/6 complex are indicated. (F) SMC complexes entrap segments of DNA to form chromatin loops and/or bridge nearby chromatin domains. Their loop extrusion activity (LE) ensures the co-entrapment of contiguously oriented intramolecular DNA segments.
Figure 6A PARP1 inhibitor treatment abolished BGL3 recruitment to DSBs. U2OS cells were treated with DMSO, PARP inhibitor (olaparib), ATM inhibitor (KU-55933), ATR inhibitor (VE-821) or caffeine. BGL3 recruitment to DNA damage sites was assessed by RNA-FISH assays. B Quantification of the positive cells. For each group, 100 randomly selected cells were counted. Data are presented as mean ±SD of three biological replicates. Two-tailed Student's t test, **P < 0.01, N: no significant difference. C PARP inhibitor mainly affected BGL3 recruitment at an early time point. U2OS cells were treated with DMSO or olaparib, and BGL3 recruitment was examined following laser micro-irradiation at indicated time point. Data presented are the average of three independent experiments, and 100 cells were counted for each experiment. Two-tailed Student's t test, **P < 0.01, N: no significant difference. D U2OS cells were transfected with the indicated siRNAs, and BGL3 recruitment to DSBs were monitored at the indicated time point by RNA FISH. Data presented are the average of three independent experiments, and 100 cells were counted for each experiment. Two-tailed Student's t test, **P < 0.01, N: no significant difference. E A model demonstrating how the BGL3 lncRNA mediates BRCA1/BARD1 complex retention at DNA damage sites.
a) HeLa cells were transfected with FLAG–TRAF6 or grown in starvation medium for 1 h.figf4(a) HeLa cells were transfected with FLAG-TRAF6 or grown in starvation medium for 1 h. Some of them were treated with chloroquine. LC3, p62 and actin were analysed by western blotting. Quantification of LC3II and p62 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.017 for LC3 and P = 0.01 and P = 0.04 for p62. (b) HeLa cells were transduced with AMBRA1 lentiviral shRNA and transfected or not with FLAG-TRAF6. Quantification of LC3II and p62 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.001 and P = 0.0018 for LC3 and P = 0.004 for p62. (c) Count of LC3 puncta in HeLa cells transduced as in b. Five different confocal microscopy images for each experiment were randomly chosen and GFP-positive dots were examined on the images with identical brightness and contrast settings. The results are represented as the average number of LC3 dots with 100 cells analysed per sample. Confocal images are shown in Supplementary Fig. S3b. (d) HEK293 cells were transfected with AMBRA1 and FLAG-TRAF6 as indicated. Protein extracts were immunoprecipitated using an anti-FLAG antibody. (e) HeLa cells were transfected as in b and FLAG-TRAF6 was purified as in d. Quantification of immunoprecipitated TRAF6 is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.0003. (f) In HeLa cells, TRAF6 was downregulated by RNAi. AMBRA1, ULK1, TRAF6, ATG13 and actin were analysed by western blotting. (g) HeLa cells were co-transfected with vectors encoding ULK1-Myc, FLAG-TRAF6 and Ub-Lys-63-HA, as indicated. Protein extracts were immunoprecipitated using an anti-Myc antibody. (h) HeLa cells stably interfered for AMBRA1 were transfected with FLAG-TRAF6 and Ub-Lys-63-HA as indicated. Protein extracts were immunoprecipitated using anti-ULK1 antibody. (i) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected as in b. The resulting precipitates were subjected to a ULK1 kinase assay using MBP as the substrate. Purified complexes were analysed by autoradiography and by western blotting using an anti-ULK1 antibody.
TIMELESS-TIPIN stimulates the priming and elongation of ubiquitin chains on CMG-MCM-7 by stabilising the association of CUL-2LRR-1 with the C.Figure 3Reconstituted CMG-MCM-7 ubiquitylation reactions were performed as above in the presence of the indicated factors. 'Neddylation' indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8. The indicated factors were assembled in the absence of ubiquitin and then incubated as for (A) before isolation of CMG via immunoprecipitation of SLD-5. The association of CUL-2 with the CMG-replisome was monitored by immunoblotting. In reactions analogous to those in (A), ubiquitylation of the indicated factors was monitored by immunoblotting. Reactions were performed in sets of three as indicated (1 = dropout of CUL-2LRR-1; 2 = wt Ubiquitin; 3 = lysine-free or K0 ubiquitin).
Figure 4(A) Namalwa cells ectopically expressing either Syndecan 1 or 4 were exposed to either SARS-CoV-2 pseudovirus alone or SARS-Cov-2 pseudovirus pre-treated with UF heparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. Binding was measured after 4 hours at 4°C by ELISA. (B) SARS-CoV-2 isolate (hCoV-19/Italy) was pre-incubated with LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. Namalwa cells expressing Syndecan 1 and 4 were exposed to either SARS-CoV-2 isolate (100 TCID/mL) or SARS-CoV-2 isolate (100 TCID/mL) pre-treated with LMWH LMWH enoxaparin (250 IU/mL) for 4 hours at 4°C and binding was determined by quantitative real-time PCR. Data show the mean values and error bars are the SEM. Statistical analysis was performed using (A) 2way-ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01 (n = 7), (B) 2way-ANOVA with Sidak's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (n = 3 measured in triplicate).
Ref(2)P analyses confirm that Dcp-1 is required for autophagic flux in ovaries.Figure 3.(A) Nondegenerating midstage egg chambers from fed w1118flies showed Ref(2) P staining in follicle cells and nurse cells. (B) Starved w1118flies contained degenerating midstage egg chambers with reduced Ref(2) P.(D) Starved Atg7flies showed an accumulation of Ref(2) P in the follicle cells and nurse cells of degenerating midstage egg chambers. (E) Starved Dcp-1Prev1flies contained increased levels of Ref(2) P in degenerating midstage egg chambers. (C) A representative Western blot of ovaries from w1118 and Atg7d77/d14 flies subjected to fed or starvation conditions for 4 d. Ref(2) P was detected by immunoblotting. Actin served as a loading control. Densitometry was performed to quantitate Ref(2) P protein levels relative to actin. Graph represents ± SD from five independent experiments (n = 5). Statistical significance was determined using a two-tailed Student's t test. *, P = 0.004 for fed samples; *, P = 0.008 for starved samples. (F) A representative Western blot of ovaries from w1118 and Dcp-1Prev1 flies that were subjected to fed or starvation conditions for 4 d. Ref(2) P was detected by immunoblotting. Actin served as a loading control. Densitometry was performed to quantitate Ref(2) P protein levels relative to actin. Graph represents ± SD from five independent experiments (n = 5)
a) Intracellular spermidine of five-day-old Δspe1 cells, compared with wild-type cells.figf2(a) Intracellular spermidine of five-day-oldΔspe1 cells, compared with wild-type cells. Data represent means ± s.e.m. (n = 3; *P 0.001). (b) Chronological ageing of wild-type (▪, o) and polyamine-depleted Δspe1 (▴, ▿) yeast cells with (open symbols) and without (closed symbols) supplementation of low doses of spermidine. Data represent means ± s.e.m. (n = 4). Cells were tested for cell death markers at day 3. (c) Fluorescence microscopy of DHE→Eth conversion in wild-type and Δspe1 cells indicating ROS production. Scale bars, 10 μm. (d) Quantification (FACS analysis) of ROS accumulation using DHE→Eth conversion of wild-type (WT) and Δspe1 cells with and without supplementation of low doses of spermidine. Data represent means ± s.e.m. (n = 4; *P 0.001). (e) Quantification (FACS analysis) of phosphatidylserine externalization and loss of membrane integrity using annexin V/PI co-staining (at day 5) and of DNA fragmentation using TUNEL staining (at day 3) of chronologically ageing wild-type (WT) and Δspe1 cells with or without supplementation of 0.1 mM spermidine as indicated. Data represent means ± s.e.m. (n = 3; *P 0.001).
Figure 7A. Schematic design of the chronic BCAA feeding experiments (n = 6 in each group). B. Body weight gain in response to the different levels of dietary BCAAs. C. Representative images of gross anatomy. D. The total weight of the extramuscular fat (posterior subcutaneous fat, epididymal fat, mesenteric fat, and retroperitoneal fat) in the mice fed high-fat diet (HFD) with or without BCAAs. E. The serum BCAA levels in the mice fed HFD with or without BCAAs. F. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon are shown. The body weight gain data are presented as a percentage of the initial body weight. The fat weight data are presented as a percentage of the body weight. G. Serum and hindlimb muscle ketone body levels, quantified enzymatically.
MCL‐1 levels regulate autophagy.Figure 3(A) H1299 cells stably expressing GFP-LC3 were infected with 10 p.f.u. per cell of either Ad LacZ or Ad Myc‐MCL‐1 for 24 h then incubated in EBSS for 4 h. Alternatively, cells were transfected with control or MCL‐1 siRNA 24 h before EBSS treatment. Cells were stained with an antibody against cyt c and analysed by immunofluorescence. Representative images are shown. Scale bars=50 μm. (B) The percent of the cell surface area covered by GFP-LC3 vesicles was quantified using the Image J software. n>70 cells in three experiment, *P0.01. (C, D) H1299 cells stably expressing GFP-LC3 were transfected as in A and incubated with 200 nM bafilomycin for 1 h where indicated. The levels of endogenous LC3‐II relative to actin were quantified by western blot in C. Data are expressed as the average of three experiments±s.d, *P0.05. (E, F) H1299 cells stably expressing GFP-LC3 were treated as in A and analysed by western blot for the presence of p62 and GFP-LC3. (G) Primary cortical neurons of the indicated genotypes were infected with a lentivirus expressing Cre at the time of plating and analysed for the presence of MCL‐1 and p62 by western blot 3 days later. (H) Transformed wild‐type (WT) and MCL‐1Δ/Δ (KO) MEFs were analysed by western blot for the expression of endogenous LC3. (I, J) Colocalization of GFP-LC3 vesicles with the lysosomal marker LAMP1 was analysed by immunofluorescence in cells treated as in A. The number of LC3‐positive vesicles that colocalised with LAMP1 was quantified in I. Data are expressed as the percentage of LC3 vesicles per cell that are LAMP1‐positive±s.e.m., with at least 14 cells per condition. Representative images are shown in J. Arrows indicate LC3‐positive vesicles colocalizing with LAMP1. Scale bars=10 μm.