EMBO/sd-character-level-ner huggingface dataset:
Figure 2(A) Proteomic profiles of different proteins, showing the relative abundance distribution across the 3x3 subfractions obtained from Jurkat cells. Proteins with very similar profiles (represented by the same colours) are likely part of the same EV subtypes. Each profile consists of three independent data triplets (F1- F2- F3A, F1- F2- F3B and F1-F2-F3C). (B) Abundance profiles of over 3000 proteins in EVs recovered after 10K (F1), 30K (F2) and 100K (F3) centrifugations , from Jurkat cells were subjected to Principal Component Analysis (PCA). Each scatter point represents one protein; proximity indicates similar profiles and hence similar distributions across EVs. The PCA scores plot was annotated with known marker proteins of intracellular organelles, and with markers as specific for CD9- or CD63- or CD81-bearing sEVs in human dendritic cells Non-marker proteins are shown as grey dots. EVs of different subcellular origin are clearly separated by the profiling analysis. PC1 and PC2 account for 44.5% and 19.1% of the variability in the data, respectively. (C) Violin plots showing enrichment of the protein markers of intracellular organelles across the F1-F2-F3 subfractions as compared to expression in the total cell proteome. Solid horizontal lines indicate medians and dashed line indicate quartiles (n=3). Mitochondria and ER markers are de-enriched, and plasma membrane markers are enriched in EV fractions, with progressively pronounced effects from F1 to F3. (D,E) Neighbourhood Network plots (top 25% quantile edges) for single queries CD63 (E, **network members, cut-off for replicates =3) and CD81 (D, *network members, cut-off for replicates =2), show several ESCRT components in the CD63 network, and ARRDC1 and integrins ITGA4/B1 in the CD81 network. A multi-query network for these two proteins and CD3G shows that all three networks are separated, indicating presence of the markers in different EV subtypes. Nodes: red = query, orange = close neighbour in all three replicates, grey = close neighbour in two out of three replicates. Edges: percentile within the local distance distribution (thicker edge and darker shade = smaller distance, i.e. closer neighbour)
Fig.Figure 6Co-culture experiments. (A) Schematic for the experimental procedure. Glia derived from mouse VM were transduced with sh-Sgk1 (B) or SGK1 (CMV-Sgk1, C), and co-cultured with VM-derived mDA neurons. Seven days after co-culture, cells were exposed to H2O2 (500µM, 3hr) or not (-), and then mDA neuron viability (TH+ cell counts) and neurite degeneration (TH+ fiber lengths) were estimated. Data are represented as mean ± SEM; n=4 culture coverslips. Student's t-test. Significantly different at p=0.033*, 0.0412**, 7.44E-23#,1.39E-23## in graph B and p=0.0489*, 0.0366**, 0.0078## in graph C. Glial neurotrophic functions potentiated by SGK1 downregulation were mediated in a paracrine manner. VM-glia transduced with sh-Sgk1 (or sh-cont as a control) were challenged with H2O2+LPS for 3hr or not. Glial conditioned medium (GCM) was prepared in each glial culture condition and administered to mDA neurons primarily cultured from mouse VM (D). Glial neurotrophic functions potentiated by SGK1 downregulation were mediated in a paracrine manner. VM-glia transduced with sh-Sgk1 (or sh-cont as a control) were challenged with H2O2+LPS for 3hr or not. Glial conditioned medium (GCM) was prepared in each glial culture condition and administered to mDA neurons primarily cultured from mouse VM (E) Cell viability of the neuronal cells was estimated using CCK8 assays and TuJ1+ cell counts. Data are represented as mean ± SEM. n=3 cultures; One-way ANOVA. Significantly different at p=0.0002*, 3.29E-08**, 0.0003***, 3.66E-06#, 0.0029## (TuJ1+ cell counting), 0.0127+, 5.98E-06++, 0.0002+++, 0.0005&, 0.0078&& (CCK8 assays) in graph E. F, Effect of the SGK1 inhibitor treatment in the mixed mDA neuron + VM-glia co-cultures. Data are represented as mean ± SEM. n=3. Student's t-test. Significantly different at p=0.0068*, 0.0368**, 0.0249#, 1.48E-27## in graph F.
Figure 7EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. Mean clinical scores of EAE (n= 5 mice per group ). Arrows indicate DC vaccine administration. EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. Effects of therapeutic DC vaccination on B6 EAE. The maximum score: Mean of the maximum scores per mouse in each group. Data shown are the mean ±SD. Determination of statistical differences was performed using by an one-way ANOVA test. EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. Recall proliferative to MOG (35-55) in splenocytes taken from DCs-treated mice 26 days after EAE induction. EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. cytokine response (D) to MOG (35-55) in splenocytes taken from DCs-treated mice 26 days after EAE induction.
Comparison of structural preservation in cryosections picked up with sucrose (a) and MU (b).Figure 1Comparison of structural preservation in cryosections picked up with sucrose (a) and MU (b). In isolated hepatocytes, autophagy was induced for 30 min in suspension buffer before fixation. (a) Cryosection picked up with 2.3 M sucrose and labeled for SOD. Note the presence of large, "empty" gaps between sequestered contents of the AV and the surrounding cytoplasm. (b) A similar section as a, but picked up with an MU mixture. The AV are no longer distorted and the overall membranous structures are sharply delineated. The SOD-labeling density of AVi is as that of the cytosol, whereas AVd show much more intense labeling. (Inset) An AVi/d that is distinct from AVi by its single limiting membrane. The highly pleiomorphic AVd are also bound by a distinctive single membrane, but in contrast to AVi/d, their contents are characterized by debris of digested material. Bars, 250 nm.
VHHASC and VHHmASC prevent IL-1β release and ASC speck formation induced by PFO, but not nigericin in primary human and mouse cells.Figure 3A-D (A-B) Human IL-1β (hIL-1β) concentrations in cell-free supernatants of LPS-primed (10 ng ml−1, 150 min) primary human macrophages that were left untreated, or pre-incubated with VHHASC or mutVHHASC (100 µg ml−1), CRID3 (50 µM) or VX-765 (50 µM) for 30 min before stimulation with (A) nigericin (10 µM), or (B) PFO (30 ng ml−1) for 2h. (C-D) Mouse IL-1β (mIL-1β) concentrations in cell-free supernatants of LPS-primed mouse BMDMs (200 ng ml−1, 150 min), incubated with VHHs, CRID3 or VX765, before activation with nigericin (10 µM), or PFO (250 ng ml−1). Data is combined from two independent experiments, each performed with two donors (A-B) or mice (C-D), represented with individual symbols (4 donors or mice in total). Data is displayed as floating bars with the max/min values and mean (thicker band). E, F (E) Epifluorescence microscopy imaging and (F) quantification of ASC speck formation in BMDMs from ASC-mCitrine (Green) transgenic mice. Cells were primed with LPS (200 ng ml−1, 150 min), pre-treated with VX-765 (50 µM, 30 min), then treated with VHHASC, VHHmASC (100 µg ml−1) or CRID3 (50 µM) for another 30 min before stimulation with nigericin (top), or PFO (bottom) for 2h and finally fixed with 4% PFA and nuclei stained with DRAQ5 (Blue). Scale bars: 100 μm. Images in (E) are representative of one out of three independent experiments that were quantified in F. Data is displayed as floating bars with the max/min values and mean (thicker band).
Induced generation of daughter cells with S-LPS.Figure 7Distribution of R-LPS and S-LPS in Δgmd plac-gmd after 3, 6, 9 and 24 h post-induction analyzed by flow cytometry (one representative example among 3 biological replicates, n = 20,000 events). Quantification of S-LPS labeling from (A) as relative frequencies. Error bars correspond to s.d. from 3 independent experiments. n0h = 77 bacteria, n3h = 254 bacteria, n6h = 616 bacteria, n9h = 937 bacteria, n24h = 1059 bacteria.
a–c, Starved LAMP1–YFP NRK cells stained with Lysotracker or DQ-BSA.figf2a-c, Starved LAMP1-YFP NRK cells stained with Lysotracker or DQ-BSA. c, Right, TEM of purified lysosomes. d, Magnification (4×) of images shown in c. Arrows indicate reformation tubules and arrowhead indicates autolysosome. e, f, Immunofluorescence (e) and immuno-EM (f) using LAMP1 antibody of starved LAMP1-YFPNRKcells shown in c. g, Density gradient fractions of NRK cells starved for times shown and blotted for LAMP2 or Cathepsin D. Middle panel shows 8-h fractions 7 and 9 analysed by TEM. Fraction 10  is the  gradient top. h, Starved LAMP1-PAGFP NRK cells were photo-activated (4 h) and imaged at 12 h with Lysotracker (upper) or DQ-BSA (lower). Boxes show enlargements. R(r) = Pearson's coefficient. Scale bars, 5 µm.
Figure 4A) Heatmap shows log2FC based alterations between the significantly different inflammation related proteins on Day 70 vs Day 0 in the CMA and placebo groups. Asterisks indicate statistical significance based on paired Student's t test. p < 0.05; B) Heatmap shows the correlation between the plasma levels of all inflammation related proteins and plasma levels of the individual metabolic activators including serine, cysteine, carnitine and nicotinamide. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Heatmap shows the associations between the significantly different inflammation related proteins (CD8A, CSF-1, CCL23, FGF-21 and OSM) Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Heatmap shows the associations between the significantly different inflammation related proteins (CD8A, CSF-1, CCL23, FGF-21 and OSM) C) with the 10 most significantly correlated plasma metabolites, D) with the abundance of the species in the gut microbiome Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Cor.Coeff: Correlation coefficient; Log2FC: log2(fold change). with the abundance of the species E) with the abundance of the species in the oral microbiome. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Cor.Coeff: Correlation coefficient; Log2FC: log2(fold change).
Constitutive expression of alternative MPC complexes A Constitutive expression of either MPCFERM or MPCOX can rescue the slow growth phenotype of the mpc1∆mpc2∆mpc3∆ triple deletion mutant.Figure 2A Constitutive expression of either MPCFERM or MPCOX can rescue the slow growth phenotype of the mpc1Δmpc2Δmpc3Δ triple deletion mutant. Growth tests were performed with cells transformed with MPC1, MPC2, or MPC3 expression plasmids or with empty vectors in indicated combinations. A serial dilution of yeast cells was spotted on agar plates with glucose‐containing synthetic minimal medium with (SD+AA) or without (SD−AA) amino acids. B Plasmid‐encoded MPC proteins can be detected in isolated mitochondria when Mpc1 is co‐expressed with either Mpc2 or Mpc3. Cells were grown in selective medium containing glycerol as a carbon source. MPC subunits were constitutively expressed from plasmids in indicated combinations in the mpc1Δmpc2Δmpc3Δ background and detected by Western blot using anti‐HA or anti‐Flag antibodies. Mitochondrial enzymes aconitase (Aco1) and malate dehydrogenase (Mdh1) were used as loading controls. C BN‐PAGE confirms the formation of MPCFERM and MPCOX complexes after constitutive plasmid‐based protein expression. Non‐specific reactivity of the antibody is denoted by an asterisk (*). It has to be noted that all complexes migrate slightly faster than in Fig C, most likely because MPC proteins are tagged with a single epitope tag as opposed to the 3HA‐tag in Fig C. D Mpc1 is co‐immunoprecipitated with Mpc2 or Mpc3. Mitochondria isolated from cells harboring appropriate expression plasmids and grown in glycerol‐containing medium were lysed in 1% digitonin, and either Mpc2‐Flag or Mpc3‐Flag was immunoprecipitated. Mpc1‐HA was detected by Western blot with an antibody directed against the epitope tag. The matrix protein aconitase (Aco1) and 40‐kDa translocase of the outer membrane subunit (Tom40) were not co‐immunoprecipitated. E, F Stoichiometry of Mpc1 complexes. Chemical cross‐linking indicates that Mpc1/Mpc2 and Mpc1/Mpc3 form heterodimers of the predicted molecular weight. DSG, disuccimidyl glutarate. Cross‐linking products with unknown partners are labeled with a question mark (?).
Figure 7(A) Example of CD3-positive cells in an olfactory bulb from an LV::S i.m.-i.n. vaccinated and protected mice and representative results from this group versus sham-vaccinated and unprotected mice at 3 dpi (n = 7-9/group). Scale bar: 50 μm. Statistical significance was evaluated by Mann-Whitney test (**= p <0.01). Cytometric analysis of cells extracted from pooled olfactory bulbs from the same groups. Cytometric analysis of cells extracted from pooled olfactory bulbs from the same groups. (C, D) Innate immune cells in the olfactory bulbs (C) or brain (D). The CD11b+ Ly6C+ Ly6G+ population in the olfactory bulbs are neutrophils and the CD11b+ Ly6C+ Ly6G- cells of the brain are inflammatory monocytes. (E) Brain H&E histology at 3 dpi. The top right and both bottom panels show examples, in two different mice, of leukocyte clusters (arrows) alongside the ventricular wall. No such clusters were detected in the LV::S i.m.-i.n. vaccinated mice (top left panel). Scale bar: 200 μm. The close up view (bottom right panel) highlights the thickened, disorganized ependymal lining, compared to the normal ependymal cells and cilia of an LV::S i.m.-i.n. vaccinated mouse (top left panel). Scale bar: 50 μm.
a) Wild-type Gli+/+ or Gli1−/−, Gli2−/− and Gli3−/− MEFs were treated with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated.f4(a) Wild-type Gli+/+ or Gli1−/−, Gli2−/− and Gli3−/− MEFs were treated with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti-actin antibodies. (b) Gli2−/− MEFs were transfected for 48 h with either empty vector or Gli2 DNA construct and treated as in a. with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti-actin antibodies. (c) LC3-II levels were detected by western blotting of total lysates from wild-type and Gli2−/− mice embryos (E12.5). For quantification, LC3-II levels from a total of five wild-type and five Gli2−/− embryos were normalized to actin, wild-type was set to 100. Graphs show mean values and error bars represent s.d. Statistical analysis were performed by two-tail Student's t-test **P0.01. Pmph, purmorphamine.
Figure 4Immunofluorescence analysis with anti-NeuN antibody in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. RGCs are preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. Scale bars are 50μm. Number of RGCs/mm (indicated as %) at one- and two-weeks post-injection. N=10. NADH dehydrogenase histochemical reaction on retinal sections of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. At one week post-injection, NADH dehydrogenase activity is lost in RGCs (GCL, areas within dashed lines) of miR-181a/b-1+/+ Rotenone-injected eyes, while it is preserved in those of miR-181a/b-1-/- Rotenone-injected eyes. GCL, Ganglion Cells Layer ONL; INL, Inner Nuclear Layer; Outer Nuclear Layer. Scale bars are 50μm. Immunofluorescence analysis, with an anti-CoxIV antibody, in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice, injected with either Rotenone or DMSO, showed preserved mitochondria in miR-181a/b-1-/- rotenone-injected eyes at one-week post-injection. Dashed boxes indicate the area of magnifications shown in right panels. Scale bars are 10μm. Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles/degree. Visual acuity is preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. N=10.
Figure 4Heatmap of ATF4-dependent mTORC1 targets (AA synthesis, 1C metabolism, AA transport, tRNA charging), ATF4 targets, and ATF4-independent mTORC1 targets (HIF1a, mt tRNA). Representative IF images showing the expression of secretory cell marker CC10, mTORC1 activity p-S6, and DATP marker CLDN4 in Scgb1a1 lineage-labeled toTomato+ cells from serial sections of control and Lats1/2 dKO lung tissues. Tomato (for Scgb1a1 lineage, red), CC10 (green), p-S6 (white), CLDN4 (magenta), and DAPI (blue). Scale bars, 100μm. Representative IF images showing the nuclear expression of YAP and ATF4 in Scgb1a1 lineage-labeled tdTomato+ cells from serial sections of control and Lats1/2 dKO lung tissues. Of note, CLDN4 expression in lineage-labeled cells having the nuclear YAP and TAZ. Tomato (for Scgb1a1 lineage, red), YAP (green), ATF4 (white), CLDN4 (magenta) and DAPI (blue). Scale bars, 100μm. Experimental designs for lineage-tracing of control, Lats1/2 double knockout (dKO) and Raptor;Lats1/2 homozygous triple-knockout (tKO) lungs. Specific time points for tamoxifen treatment and analysis are indicated. Representative H&E and IF images of control, Lats1/2 dKO, and Raptor;Lats1/2 tKO airways at day 21 post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green, upper), AGER (green, lower), and DAPI (blue). Scale bar, 100μm. Quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells (left) and tdTomato+AGER+ AT1 cells (right) in (E). Data are presented as mean ± SEM (n=2-3 mice for each genotype). ***p<0.001 (Student's t-test). Quantitative PCR (qPCR) analysis of ATF4-dependent amino acid transporters (Slc7a1, Slc7a3, Slc7a5, Slc34a2, Slc7a11), on freshly isolated Scgb1a1 lineage-labeled cells from control and Lats1/2 dKO lungs at day 2 post tamoxifen treatment. Data are presented as mean ± SEM (n=2 mice for each group). *p<0.05, ***p<0.0001 (Student's t-test). Measurement of L-amino acid or essential amino acids (leucine, isoleucine, valine) contents in Scgb1a1 lineage-labeled tdTomato+ organoids derived from control, Lats1/2 dKO or Raptor;Lats1/2 tKO mice. Data are presented as mean ± SEM (n=3 technical replicates for each genotype). *p<0.05, **p<0.001 (Student's t-test). Representative IF images showing the expression of YAP, p-S6, and ATF4 in serial sections of 3D organoids derived from secretory cells in indicated culture conditions. Tomato (for Scgb1a1 lineage, red), YAP (green), p-S6 (white), ATF4 (magenta), and DAPI (blue). Scale bars, 100μm.
Figure 4HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted (KD) of Stx17, PGAM5 or Drp1 were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies. HeLa cells stably expressing GFP-Parkin with mock treatment (Mock) or depleted of Stx17, PGAM5 or Drp1 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by immunofluorescence microscopy. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of GFP-Parkin and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test). PINK1-FLAG and GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (ethanol) or presence of 20 μM CCCP for 2 h and analyzed by IB using the indicated antibodies. F, full-length PINK1; C, cleaved PINK1.
Figure 4(A, B) WT (Pml+/+) and Pml-/- mice (n = 12 in each group) were injected with mouse TNF (1.5 μg/g body weight), and rectal body temperature (A) and survival (B) were determined at the indicated time-points. #, the temperature of only six mice was measured at the indicated time-points. Mean ± S.E.M is shown. *P < 0.05 as determined by two-way ANOVA (A), or by Log-rank (Mantel-Cox) test (B). (C, D) WT and Pml-/- mice were injected with TNF and serum samples from live mice were collected 30 h later. Levels of LDH (C) and GPT/ALT (D) were quantitated. Mean ± S.E.M. is shown. P = 0.0095 (C, D) by unpaired t-test with Welch's correction. Serum levels of TNF (E) from WT and Pml-/- mice 12 h after TNF injection. Serum levels of IL-6 (F) and IL-1α (G) from WT and Pml-/- mice 12 h after TNF injection.
Figure 5A HeLa cell line stably expressing Mad1-BirA was depleted of Rod and synchronized in mitosis using thymidine and nocodazole. Biotin was added to media where indicated and biotinylated proteins were purified and analyzed by western blot with the indicated antibodies. Representative of 2 independent experiments. The duration from NEBD to exit in each indicated conditions was recorded by time-lapse microscopy. Mitotic duration in the indicated conditions expressing Mad1-Bub1 (Mad1 485-718 fused to Bub1 1-553ΔCD1) fusion protein. The time from NEBD to exit is indicated. Red circles represent cells still arrested in mitosis when the filming stopped. Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs. (D) Representative immunofluorescence images of cells stained for YFP and Mad1 in each condition. Scale bar, 5 μm. Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs. Mad1 kinetochore levels were measured and normalized to Bub1 (YFP) level. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.