EMBO/sd-character-level-ner huggingface dataset:
Targeting the Asm system prevents melanoma metastasis.Figure 8A Amitriptyline (2 mg/kg, Ami) was intraperitoneally injected into C57BL/6mice for five times every 12 h. Sixty minutes after the last injection, 1 × 105B16F10tumor cells were intravenously injected. Control experiments confirmed that amitriptyline inhibited Asm activity in the blood by approximately 85%. Asm heterozygous mice were injected with 1 × 105B16F10tumor cells. The number of lungmetastases was determined after 14 days. Shown is the mean ± SD from six mice each, ANOVA followed by Tukey's test for multiple comparison. P-values are indicated. B, C MT/ret cells (105) suspended in 200 μl Matrigel/PBS (1:1) were injected s.c. in the left and right flanks of 8-week-old Asm-deficient mice or wild-type mice. Mice were sacrificed at day 20 after injection, and spleens were inspected for the presence of metastases. The representative photographs (B) show the results from one of four experiments, and the quantitative analysis is displayed in (C). B, C MT/ret cells (105) suspended in 200 μl Matrigel/PBS (1:1) were injected s.c. in the left and right flanks of 8-week-old Asm-deficient mice or wild-type mice. Mice were sacrificed at day 20 after injection, and spleens were inspected for the presence of metastases. The representative photographs (B) show the results from one of four experiments, and the quantitative analysis is displayed in (C). D Platelets were isolated from wild-type or Asm-deficient mice by density-gradient centrifugation, and 1 × 107 platelets were incubated with 1 × 105 MT/ret melanoma cells. The samples were lysed, and Asm activity in cell lysates was determined. In unstimulated samples (time point 0 of co-incubation), tumorcell and platelet lysates were admixed after lysis. E To measure secretion of Asm by platelets, tumor cells and platelets were co-incubated for 30 s, the samples were pelleted, the supernatants were acidified, and the Asm activity was measured. All Asm activity measurements were performed in the presence of 100 μM Zn2+. F Local tumor growth in wild-type or Asm-deficient mice at the flank was measured at day 20.
a) Diagram illustrating how lysosomal positioning coordinates mTOR signalling and autophagy.figf5(a) Diagram illustrating how lysosomal positioning coordinates mTOR signalling and autophagy. Peripheral lysosomal localization (nutrient induced) increases mTOR activity (blocking autophagosome synthesis) and reduces autophagosome-lysosome fusion. Starvation-induced lysosomal clustering reduces mTOR activity (activating autophagosome synthesis) and facilitates autophagosome-lysosome fusion. (b) Serum and amino-acid starvation of HeLa cells for 5 h reduces LC3-II levels (above), but increases autolysosome numbers detected using tfLC3 (below). With tfLC3, GFP- and RFP-positive puncta represent autophagosomes before lysosomal fusion, whereas RFP-positive/GFP-negative puncta represent autolysosomes-GFP is more rapidly quenched by low lysosomal pH (see Methods). Increased autolysosomes indicate enhanced starvation-induced flux of LC3 to lysosomes. (c) ARL8B knockdown increases autophagosomal synthesis. siRNA-transfected HeLa cells were incubated for 48 h, then left untreated or incubated with bafilomycin A1. LC3-II levels versus actin were quantified (bottom graphs). Asterisk: nonspecific band. (d) ARL8B overexpression inhibits autophagosome synthesis and degradation. HeLa cells overexpressing ARL8B or empty vector were analysed as in (c). (e) HeLa cells were co-transfected with either ARL8B overexpression construct or siRNA (non-targeting siRNA and empty peGFP vector were transfection controls) together with mCherry-LC3 for 48 h. After fixation, cells were stained for endogenous LAMP1 and DNA (DAPI). Representative maximum-intensity projections of serial confocal optical sections are shown. Co-localization panels show overlapping mCherry-LC3 and LAMP1 signals. (f-h) Quantification of autophagosome-lysosome fusion in HeLa cells. Percentages of autolysosomes (positive for both mCherry-LC3 and LAMP1) to autophagosomes (positive for mCherry-LC3 and negative for LAMP1) were quantified. In (h), we analysed cells treated for 2 h with nocodazole before fixation, which dispersed the perinuclear lysosomal cluster (see Fig. 2a). (i) Autophagosomal and autolysosomal numbers in tfLC3-expressing cells after ARL8B overexpression and knockdown (Supplementary Fig. S5o shows representative cells.). In (f-i) we analysed 20 cells per group in three independent experiments. Values are means ± s.e.m of three independent experiments carried out in triplicate. *P0.05, **P0.01, ***P0.005 Student's t-test; other comparisons not significant (n.s.). Uncropped images of blots are shown in Supplementary Fig. S7.
Comparison of SARS-CoV-2 and SARS-CoV replication kinetics in Vero E6 cells.Fig. 2. Comparison of SARS-CoV-2 and SARS-CoV replication kinetics in Vero E6 cells.(a) Growth curve showing the release of infectious viral progeny into the medium of infected Vero E6 cells (m.o.i. 3), as determined by plaque assay (n = 4; mean ± sd is presented). (b). Comparison of SARS-CoV-2 Australia/VIC01/2020 and SARS-CoV Frankfurt-1 plaque phenotype in Vero E6 cells.
Physiological role of the interaction between ALFY and GABARAP.Figure 4A HeLa FlpIn GFP−GABARAP cells were treated with proteasomal inhibitor (MG132, 2 h) or subjected to amino acid starvation (EBSS, 2 h), before staining with anti−ALFY antibodies. Scale bar, 10 μm. B GFP−tagged full−length ALFY wild−type or LC3−interacting region (LIR)−mutant was expressed into Alfy−deficient MEFs using an adenovirus system. At 48 h after infection, the MEFs were cultured in normal media or EBSS for 1.5 h before staining with anti−LC3B or anti−GABARAP antibodies. Scale bars 10 μm. C HeLa cells were treated with control or siRNA targeting GABARAP, GABARAPL1 and GABARAPL2. 72 h after transfection cells were immunostained with anti−LC3B, anti−ALFY and anti−p62 antibodies. Scale bars, 10 μm. D HeLa cells were treated with control or siRNA targeting ALFY. 72 h after transfection, cells were treated with puromycin with or without bafilomycin A1 for 2 h and total cell lysates were fractionated into TX−100−soluble and insoluble fractions. The TX−100−soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments. E ALFY WT and KO MEFs were treated with puromycin or EBSS with or without bafilomycin A1 for 2 h and the total cell lysates were then fractionated into Triton X−100 (TX−100)−soluble and insoluble fractions. The TX−100−soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments. F Schematic model of ALFY‐mediated selective autophagy.
A.Figure 6A. Intracellular tachyzoites expressing GFP-TgAtg8 were used to invade host HFF and the proportions of cells displaying punctate or cytosolic GFP signals were assessed along time during development. Data are mean from n = 5 independent experiments ±SEM. B. Fluorescence microscopy pictures of GFP-TgAtg8-expressing tachyzoites during their intracellular development and division. The arrowhead indicates the residual body made of maternal material left after endodyogeny. C. Detection of GFP-TgAtg8-labelled autophagosomes in dividing parasites. Progression of daughter cells division was followed by expression of an IMC1-RFP construct.
Figure 7Working models for histone Kcr to regulate neural stem cell fate decisions.
HMGCL targeting limits oncogenic properties of pancreatic tumor cellsA Quantification of colony forming area of sg-CTRL and sg-HMGCL (#2, #3 and #4 clones) PANC-1 cells.Figure 3A Quantification of colony forming area of sg-CTRL and sg-HMGCL (#2, #3 and #4 clones) PANC-1 cells. Colony area is expressed as mean fold change relative to sg-CTRL ± SEM (n=3 independent experiments). Significance was defined by one-tailed Student's t-test. *** p<0.001. B Quantification of wound healing capacity of sg-CTRL and sg-HMGCL (#2, #3 and #4 clones) PANC-1 cells. Wound healing capacity is expressed as mean of percentage of restored area at 72hr ± SEM (n=4 independent experiments). Significance was defined by one-tailed Student's t test. ***p<0.001. C, Quantification of migratory sg-CTRL and sg-HMGCL (#2, #3 and #4 clones) PANC-1 cells. Migratory cell numbers are expressed as mean fold change relative to sg-CTRL cells ± SEM (n=3 independent experiments). Significance was defined by one-tailed Student's t test. ***p<0.001. D Quantification of invasive sg-CTRL and sg-HMGCL (#2, #3 and #4 clones) PANC-1 cells. invasive cell numbers are expressed as mean fold change relative to sg-CTRL cells ± SEM (n=3 independent experiments). Significance was defined by one-tailed Student's t test. ***p<0.001. E Representative images of spheroid formation from sg-CTRL and sg-HMGCL (#2, #3 and #4 clones) PANC-1 cells after 4 or 15 days of culture. Photos are representative of n=3 independent experiments. F Representative images of spheroid invasion assay from sg-CTRL and sg-HMGCL (#2, #3 and #4 clones) PANC-1 cells after 10 days of culture in presence of Matrigel. Photos are representative of n=3 independent experiments. G Volcano plot illustrating proteins significantly under- or over-represented in sg-CTRL and sg-HMGCL PANC-1 spheroids (n=3 independent experiments). Significance was defined by one-tailed Student's t-test. Protein levels with a q-value<0.05 (horizontal axis) and a fold change <-1.5 or >+1.5 (vertical axis) are considered as significantly down or up-regulated in sg-HMGCL PANC-1 spheroids.
The first 30 amino acids of TFEB are sufficient for binding to active Rag heterodimers.Figure 7.(A) ARPE-19 cells coexpressing active RagB/D heterodimer and the indicated TFEB plasmids were fixed, permeabilized with 0.2% saponin, and stained with antibodies against GST (used to detect Rag proteins). Regions within the dotted boxes are magnified in the insets. Bars, 10 µm. (B) ARPE-19 cells were cotransfected with active RagB/D heterodimers and the indicated TFEB constructs. After 18 h, cell lysates were immunoprecipitated with the anti-HA antibody (used to immunoprecipitate Rag proteins) and immunoblotted with antibodies against GFP and GST (used to detect TFEB-GFP and Rag proteins, respectively). The white line indicates that intervening lanes have been spliced out. (C) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-(1-30)-GFP in ARPE-19 cells coexpressing active RagB/D heterodimers (antibodies against GST were used to detect Rags). Bar, 4 µm. (D) Relative RT-PCR analysis of the mRNA expression of autophagy (ATG9B and UVRAG) and lysosomal (MCOLN1) genes in ARPE-19 cells infected with the indicated adenovirus for 48 h. The values are expressed as a ratio to RNA from cells infected with control adenovirus (Ad.-Null). Values are means ± SD of two independent experiments. ***, P < 0.001; **, P < 0.01. (E) Cells were infected with adenovirus as in D and analyzed by immunoblotting with the indicated antibodies. IP, immunoprecipitation.
a) Quantification of the number of ubiquitin-positive, p62-positive or GFP–LC3-positive bacteria.figf3(a) Quantification of the number of ubiquitin-positive, p62-positive or GFP-LC3-positive bacteria. MDCK/pGFP-LC3/pp62-3×Myc cells were infected with a wild-type or ΔactA2 strain of L. monocytogenes for 2 h. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 6 experiments). (b) Colocalization of p62 with ΔactA2. MDCK/pGFP-LC3/pp62-3×Myc cells were stained with an anti-ubiquitin antibody (light blue), anti-Myc antibody (red), DAPI (white) and anti-GFP-LC3 (green). Scale bar, 10 μm. Arrowheads indicate GFP-LC3-, ubiquitin- or p62-positive bacteria. (c) Quantification of the number of ubiquitin-positive, p62-positive or GFP-LC3-positive bacteria. p62−/−/p62 cells were infected with ΔactA2 bacteria for 2 h. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 6 and 3 experiments for p62−/− and p62−/−/p62, respectively) (d, e) Intracellular survival of wild-type or ΔactA2 bacteria in p62−/− cells (d) or p62−/−/p62 cells (e; colony-forming units, CFU, relative to CFU 30 min after infection). Data are presented as mean ± s.e.m. (n ≥ 4 experiments). (f) Schematic representation of p62 mutants. The deleted amino-acid regions of ΔLIR and ΔUBA are 321-342 and 394-431, respectively. ΔLIR-ΔUBA has deleted 321-342 and 394-431 amino-acid regions. K7AD69A has two amino-acid substitutions. (g) Generation of p62-3×Myc mutants in p62−/− cells. (h) Quantification of the number of GFP-positive or GFP-LC3-positive bacteria. p62−/−/p62 (wild type, ΔLIR, ΔUBA, ΔLIR-ΔUBA or K7AD69A) cells were infected with ΔactA2 bacteria and stained with anti-bacteria and anti-Myc antibodies (Supplementary Information, Fig. S4e). Data are mean ± s.e.m., at least 500 LC3 were counted in each experiment (n = 3 experiments). P 0.001 versus p62-positive LC3 in p62−/−/p62 wild-type cells, P 0.001 versus GFP-p62-positive bacteria in p62−/−/ubiquitinwild-type cells. Ub, ubiquitin. WT, wild type. A full scan of g is shown in Supplementary Information, Fig. S7.
Rab8A Ser111 phosphorylation is abolished in Parkinson's disease patient PINK1 fibroblasts and PINK1 knockout mouse embryonic fibroblasts (MEFs).Figure 5Absence of Rab8A Ser111 phosphorylation in human mutant PINK1 patient fibroblasts. Primary skinfibroblasts were derived from a patient with homozygous PINK1 Q456X mutation or unaffected control. Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with polyclonal anti‐PINK1 antibody and immunoblotted with monoclonal PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH. Absence of Rab8A Ser111 phosphorylation in PINK1 knockout MEFs. MEFs were derived from PINK1 knockout embryos or wild‐type controls (see Materials and Methods). Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with a polyclonal anti‐mouse‐specific PINK1 antibody and immunoblotted with a different anti‐mouse‐specific PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.
Determination of an optimal concentration for each HDACI to induce HIV-1 production in latently-infected cells.Figure 2A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of HDACIs. At 24 h post-treatment, viral production was measured by quantifying p24 antigen production in culture supernatants (A, B) A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of HDACIs. At 24 h post-treatment, metabolic activity was assessed by a WST-1 assay (C, D).Data information: Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value 100% (C, D). The selected doses were indicated by "an arrow". Plasmatic concentrations after usual dosage (Cmax) of each drug in human therapy are indicated below the LRA names and by a box in the graph.
AltFUS expression is necessary for the full FUS-linked ALS phenotype in Drosophila A, Crossbreeding strategy for Drosophila generation using the Elav-GeneSwitch-GAL driver as an inducible expression system specific to the motor neurons.Figure 5A, Crossbreeding strategy for Drosophila generation using the Elav-GeneSwitch-GAL driver as an inducible expression system specific to the motor neurons. B, FUS and altFUS expression in mCherry (control), altFUS, FUS, FUS(Ø), FUS-R495x or FUS(Ø)-R495x expressing Drosophila from the control F1 and the RU-486 treated F1 at 1, 10 or 20 days post-induction (representative image from n=3). C-E, Locomotion assay represented by the percentage of climbing success in control and RU-486-treated transgenic Drosophila expressing mCherry or altFUS (C), the bicistronic FUS or the monocistronic FUS(Ø) (D), and the bicistronic FUS-R495x or the monocistronic FUS(Ø)-R495x (E) at day 1, 10 and 20 post-induction. Statistical comparisons were made between each population (n=4 - biological replicates). Indicated significance is between the monocistronic and the bicistronic transgenic flies of the RU-486-treated population (mean ± SD, n.s. = non-significant, * = p value < 0.05, *** = p value < 0.001, two-way ANOVA with Tukey's multiple comparison correction).
Figure 7A-B. AAV-HDAC3 mutant (Y298H; dominant negative deacetylase inactive) promoted DRG regenerative growth after spinal cord injury vs AAV-V5 control (injected 5 weeks prior to SCI in the sciatic nerve bilaterally) as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, (A-B) 200 μm. C-F. Immunofluorescence for H3K9ac. AAV-HDAC3 mutant induced upregulation of H3K9ac in vivo in DRG neurons, but not in surrounding nuclei of satellite cells. Scale bar, (C-F) 50 μm. G-J. New synaptic formations (VGlut1+) from regrowing axons are shown beyond the lesion site after injection of AAV-HDAC3mut. (G) Scale bar 200 μm, (H-J) High magnification insets depict co-localization of Dextran and vGlut1 staining. Scale bar 50 μm. K-L. Quantification of dextran positive axons shows that AAV-HDAC3mut promotes DRG regenerative growth across and beyond the spinal lesion site. Data is expressed as percentage of dextran+ axons at each distance vs dextran+ axons at -700μm from the lesion margin (K) or distance from the caudal margin of the lesion to the most rostral dextran+ axon tip for each animal (L). N= 10 animals per condition, ± s.e.m. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 indicate a significant difference (ANOVA followed by Bonferroni test).
Figure 1A. Yeast two-hybrid assays. Yeast cells carrying different fusion protein combinations are listed on the left. Yeast cells expressing the indicated proteins from the pGAD-T7 (AD) and pGBK-T7 (BD) vectors were plated onto medium lacking Leu and Trp (SD-LT) (left) or medium lacking Leu, Trp, Ade and His (SD-LTHA) (right). B. Co-immunoprecipitation (co-IP) of IDM1 with IDAP1 or IDAP2 in transgenic plants. Transgenic plants expressing IDM1-HA-YFP and IDAP1-3Myc or IDAP2-3Flag under their native promoters and their F1 offspring were used for co-IP analysis. Input, total protein before IP. C. Detection of proteins that associate with IDAP1 or IDAP2. Proteins were detected using LC-MS/MS following IP of Myc-tagged IDAP1 or Flag-tagged IDAP2. D. The elution profiles of IDM1-HA-YFP, IDAP1-3Myc, and IDAP2-3Flag in gel filtration assays. Anti-HA, anti-Myc, and anti-Flag antibodies were used in Western blots.
NSMCE2 colocalizes with BRCA1 at sites different to DSBImmunohistochemistry of NSMCE2 on mouse testis.Figure 1Immunohistochemistry of NSMCE2 on mouse testis. The inset illustrates the accumulation of NSMCE2 on the XY body. Scale bar, 50 μm. Immunofluorescence (IF) of NSMCE2 and SCP3 on isolated mouse spermatocytes. Threads of NSMCE2 can be detected on regions of weak SCP3 signal, indicative of asynapsis (arrowheads). Scale bar, 2.5 μm. Colocalization of NSMCE2 and BRCA1 at the XY body of mouse spermatocytes. Representative image of NSMCE2 and BRCA1 foci in control and MMS‐treated MEFs. Scale bar, 2.5 μm. Quantification of NSMCE2 and BRCA1 foci in wild‐type MEFs exposed to IR (10 Gy; 1‐h recovery), MMS (1 mM for 1 h; 24‐h recovery), and PARP inhibitor (1 μM for 1 h; 24‐h recovery). Data indicate mean values and are representative of three independent experiments. Error bars indicate SD. Representative image of IR‐ (10 Gy; 1‐h recovery) and MMS‐ (1 mM for 1‐h; 24‐h recovery) induced NSMCE2 and γH2AX foci in MEFs. Scale bar, 2.5 μm.
– Mutants of cycb1 ku70 are hypersensitive to BLM: Genetic interaction between NHEJ und HR.Figure 7A. The wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 were grown on control plates and root lengths were measured 10 days after germination.B. The wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 were grown on plates containing 0,6 μg/mL BLM. Root lengths were measured 10 days after germination.C. Graph represents the ratio of the mean growth rate on 0,6 µg/mL BLM compared to control experiments on plates lacking BLM for the wildtype, ku70, cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70. D. Image shows the wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 (from left to right) grown on control plates 10 days after germination.E. Images show the wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 (from left to right) on plates containing 0,6 μg/mL BLM.
Figure 5ChIP-seq tracks of T (in red; input is in blue) and core pluripotency factors (in green) around the Blimp1 (100 kb) and Prdm14 (100 kb). The co-bound locus (L#1-4) and the locus exclusively bound by core pluripotency factors but not T (L#5) are indicated by arrow. NC, negative control (the locus without T binding). ChIP-qPCR for the enrichment of the indicated factors on the loci (L#1-5) indicated in (A). The enrichment is indicated by the percent input with SDs (three independent experiments with two technical replicates). Luciferase assay with the specific sequence of the locus bound by T around L#1 and L#3 as shown in (A). Relative luciferase activities of low T-inducible ZHBTc4 ESCs stimulated as indicated are shown with SDs. (three independent experiments with two technical replicates). Expression of Blimp1 and Prdm14 measured by qPCR in PGCLCs derived from indicated ESC lines. The ∆Ct values from the average Ct values of Arbp and Ppia are shown (log2 scale, the mean value of two independent experiments with two technical replicates). Top: Oct4-GFP (green) reporter expression (left), and Blimp1-RFP (red) and Oct4-GFP (BROG) reporter expression (right) in the gonads of E13.5 embryos generated by 4N complementation with the indicated ESC lines. Scale bar: 100um. Bottom: percentage of Oct4-GFP reporter-positive PGCs (left) and BROG reporter-positive PGCs (right) measured by FACS analysis. Blue circles, red squares, and green triangles represent the percentage of gonadal PGCs in the first, second, and third independent mutant line, respectively, of the examined locus (L#1-5). Model indicating the inductive germ cell segregation achieved by a co-regulatory mechanism of residual pluripotency regulators and lineage regulators.