EMBO/sd-character-level-ner huggingface dataset:
Formation of autolysosomes is inhibited in cells depleted of Tsg101 or Vps24.Figure 4.Formation of autolysosomes is inhibited in cells depleted of Tsg101 or Vps24. (A) The double-tagged LC3 protein (mCherry-GFP-LC3) will emit yellow (green merged with red) fluorescence in non-acidic structures and appear as red only in the autolysosomes due to quenching of GFP in these acidic structures. (B-F) HeLa cells were transfected with control (B), Tsg101 (C), or Vps24 (D) siRNA for 4 d and then with mCherry-GFP-LC3 for another 24 h before confocal microscopy analysis. Bar, 5 μm (E) The mCherry-LC3 (red) signal was quantified in ∼30 cells with similar expression levels from three independent experiments using the Zeiss LSM 510 Meta software and the data are presented as % of total mCherry-GFP-LC3. Error bar = SEM. (F) Degradation of [14C]-valine- labeled proteins in cells incubated in complete medium (control), serum- and amino acid-deficient medium (starve), or starvation media in the presence of 3-methyl adenine (3-MA) to inhibit autophagic degradation. Average degradation per hour from four experiments done in duplicates ±1 SEM are shown.
In vivo” treatment with ETP-47037 compound does not compromise tissue viability.Figure 9A-C Quantification of the number of (A) Ki67-, (B) pan-nuclear p-H3 pattern-, and (C) foci p-H3 pattern-positive cells in untreated and ETP-470037-treated lungcarcinomas. The data represent the mean values obtained for three mice in each group. Error bars represent standard errors.D Representative Ki67 and p-H3 images. E Representative H&amp;amp;E images of intestine samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown to the right indicating the presence of normal mitosis, giant multinucleated and aberrant mitotic figures. F Representative H&amp;amp;E images of bone marrow and skin samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown indicating the presence of necrosis, hemosiderosis, multinucleated cells, and giant nuclei. Bone marrow showed moderated aplasia. G Telomere length in untreated and ETP-47037-treated lung tumor samples. Representative images are shown to the right.
Figure 2A, B WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. The fold changes in mRNA levels of SNAI1 (encoding Snail) (A) and SNAI2 (encoding Slug) (B) in Caco-2 cells were determined by RT-qPCR. n = 6 independent experiments. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ns, not significant. C WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. Cytoplasmic and nuclear proteins were extracted for Snail and Slug determination by immunoblots. WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. Snail and Slug was assessed by immunoﬂuorescence staining (D) Scale bar: 20 μm. n = 3 independent experiments, n = 18 fields analysed. WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. quantiﬁed the percentage of Snail (E) or Slug (F)-positive nuclei respectively. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001. G Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without 100 μg/mL OMVs for 24 h. Proteins from nucleus, cytoplasm and total cell lysates were subjected to immunoblots with the indicated antibodies. H WT and SNX10 KO Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without 100 μg/mL OMVs for 24 h. Proteins from nucleus, cytoplasm and total cell lysates were subjected to immunoblots with the indicated antibodies.
Bioinformatics analysis of candidate autophagy-regulating genes and identification of PP2A as a complex regulator of autophagy.Figure 1.(A) All analyzed 318 siRNA target genes were categorized into phosphatase families and marked according to the ability of their corresponding siRNAs to regulate EGFP-LC3 accumulation. PIP, phosphatidylinositol phosphatase; PTP, phosphotyrosine phosphatase; DUSP, dual-specificity phosphatase; PPP, (Ser/Thr) phosphoprotein phosphatase. (B) Canonical pathways with significant enrichment of candidate genes were identified by hypergeometric method using the Ingenuity Pathway Analysis software. Blue bars indicate the −log of the p-values (Fisher's exact test) of the enrichment (left vertical axis). The orange line indicates the percentage of the identified autophagy-regulating genes of all genes in the respective pathway (right vertical axis). The threshold line indicates the position of the P = 0.05. See Table S1 for genes associated with these pathways. AMPK, AMP-activated protein kinase; ERK, extracellular signal-regulated kinase; ROS, reactive oxygen species; ILK, integrin-linked kinase; iCOS, inducible T cell co-stimulator. (C) Schematic representation of the PP2A holoenzyme that is composed of a catalytic subunit (PP2Ac), a scaffold protein (PR65), and a variable regulatory B subunit that interacts with the complex via PR65-α/β HEAT domains (Huntington/elongation/A subunit/TOR). CIP2A interacts with PP2A holoenzyme and modulates its activity toward specific substrates. The gene names for the identified autophagy-regulating candidates and the corresponding proteins (bold) are indicated. (D) Quantification of EGFP-LC3 puncta in MCF7-EGFP-LC3 cells 56 h after transfection with the indicated siRNAs. When indicated, cells were exposed to 100 nM rapamycin for the last 3 h. RPTOR (raptor) and BECN1 (beclin1) siRNAs served as positive and negative controls, respectively. (E) Autophagic flux was measured as the ratio between luciferase activities in MCF7-RLuc-LC3WT and MCF7-RLuc-LC3G120A cells transfected with the indicated siRNAs 56 h earlier and left untreated or treated with 100 nM rapamycin for the last 3 h. Error bars are SDs for a representative (n = 5) triplicate experiment with a minimum of 4 × 10 randomly chosen areas/sample analyzed (D) or three independent experiments (F). *, P < 0.05; **, P < 0.01; ***, P < 0.001, as compared with similarly treated control siRNA-transfected samples.
ER proteins are rerouted to the cytosol in human and mouse Glioblastoma (GBM) tumors.Figure 1Representative western blots after subcellular protein fractionation experiment in isolated murine-derived non-tumor (NT) and GBM tumor (T) tissues. Total levels of the ER-lumenal proteins tested from the cell lysate derived from tissues used in (A). Quantification of the protein levels of ER lumenal proteins in the cytosolic fraction as shown in (A). n = 5 biological replicates, the horizontal line represent the sample ﻿mean. ﻿Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated. P-values < 0.05 were considered significant). Human derived GBM tumors were processed as in (A). Representative western blot were performed (F) and the percentage of ER-lumenal protein cytosolic localization (G) were quantified. Data are the average from six different tumors. N=6 Bars and error bars indicate mean
Figure 5(A) Representative stereoscopic image of P6 WT and IκBα KO-derived intestinal organoids. Scale bars in A, 100 μm (B) IHC analysis with the indicated antibodies and Alcian Blue staining of WT and IκBα KO-derived organoids. Scale bars in B 50 μm. (C) qPCR analysis of the indicated ISC genes in WT and IκBα KO-derived organoids. Data information: 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, * p-value<0.05. (D) Correlation plots of the indicated differentially expressed gene (DEG) sets obtained in expression microarray (5 technical replicates of a minimum of two organoids per genotype were analyzed). Values for DEG in fetal and adult organoids were obtained from Mustata et al, 2013. (E) qPCR analysis of the indicated fetal genes in WT and IκBα KO-derived organoids. Data information: 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, * p-value<0.05. (F) Western blot analysis of control and IκBα-deleted organoids using CRISPR-Cas9 technology. (G) Representative stereoscopic image of WT and IκBα-deleted organoids. Scale bars in G, 50 μm.
Figure 6level of PIP2 in M-MOPS was examined by measuring immunofluorescence from images at set time points after exposure after exposure to 5µg/ml anti-FcγRII/III (2.4G2), 50 ng/ml LPS or oligomers of 40 µM Aβ1-42 Data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA with Sidak's multiple comparison *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522). The levels of PIP2 was also measured using a plate reader after exposure to physiologically relevant stimuli in M-MOPS and primary mouse microglia Data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA with Sidak's multiple comparison *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522). DAG level were measured using a live cell assay in M-MOPS (and primary mouse microglia as a time course from immunofluorescence Data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA with Sidak's multiple comparison *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522). plate reader after exposure to physiologically relevant stimuli Data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA with Sidak's multiple comparison *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522).
Figure 5A Bar graph showing the ROS levels in the NFIB models. The fluorescence signal was normalized to the blank. n = 2 biological replicates and n = 2 technical replicates, means ± s.d., two-tailed Student's t-test. B Left panel: representative images of HIF1α immunofluorescence. HIF1α was stained with Alexa-633 (red) and nucleus with DAPI (blue). Scale bar 100 μm. Right panel: bar graph showing quantification of HIF1α nuclear/cytoplasmatic ratio. n = 5 biological replicates, means ± s.d., two-tailed Mann-Whitney U-test. C Left panel: Bar graph representing mean VEGFA mRNA expression in SUM159PT gCTRL and SUM159PT gNFIB breast cancer cell lines upon downregulation of ERO1A with two independent shRNAs (sh1 and sh2). Right panel: Bar graph representing mean VEGFA mRNA expression in lung extracts from animals inoculated i.v. with SUM159PT gNFIB shCTRL, SUM159PT sh1 ERO1A, or SUM159PT sh2 ERO1A cells. In both graphs n = 3 biological replicates and n = 2 technical replicates, means ± s.d., two-tailed Student's t-test, FC= fold change. E Left panel: Bar graph showing average of master segments counted after incubation of HUVEC cells with conditioned medium from LM1, HR1, 4T1 (Nfib high-expression and the respective Nfib low-expression or KO controls) and SUM159PT gNFIB shCTRL, SUM159PT gNFIB sh1 ERO1A, SUM159PT gNFIB sh2 ERO1A cells. Human VEGF121(0.4 ng/μl) was added to SUM159PT gNFIB sh1 and sh2 ERO1A cells, n = 2 technical replicates, means ± s.d., two-tailed Student's t-test. Right panel: Representative images showing tube formation four hours after addition of conditioned medium from HR1 gCtrl or g4Nfib.Scale bar 100 μm.
Figure 1Representation of mice treatment IHC images for collagen The inserts in panel B represent the hematoxylin (nucleus) & eosin (cytoplasm) staining for the same tissue area of the collagen staining In all cases n=4 control mice, and ≥3 mice for 2mg and 5 mg) The quantification for each staining is shown in the histogram in the right side IHC images fo aSMA, CD68, and CD204 For aSMA, CD68, and CD204 the percentage of positive cells is relative to the control condition. In all cases n=4 control mice, and ≥3 mice for 2mg and 5 mg) Scale bar: 100 mm. The quantification for each staining is shown in the histogram in the right side
Membrane binding by Atg5 is essential for autophagy and the Cvt pathway in vivo.Figure 7(A) Liposome co‐sedimentation assay using Folch liposomes and the indicated Atg5 proteins stabilized with an N‐terminal fragment of Atg16 (amino acids 1-46). (B) Quantification based on three co‐sedimentation assays as shown in (A). The amount of protein pelleted in the absence of liposomes was subtracted. The averages and the standard deviations are shown. (C) Yeast cells were transformed with the indicated expression constructs and Atg5-2 × Myc was immunoprecipitated with an anti‐Myc antibody. The precipitated protein was subjected to anti‐Atg5 and anti‐Atg12western blotting. The substitution of R171 and K160 to glutamate caused the mutant protein to run higher. The same phenomenon was observed for the recombinant protein ( Supplementary Figure 3). (D) Anti‐Atg12 western blot of cell fractions prepared from rapamycin‐treated yeast cells. The anti‐Pgk1 and anti‐Pex30 served as control for the cytosolic and membrane fractions, respectively. (E) Anti‐Myc and anti‐Pex30western blots of yeast cell fractions from cells of the indicated genotype and expressing Atg16 and Atg5 or Atg5 K160E, R171E. (F) Starvation assay with the indicated yeast strains and the indicated rescue constructs. GFP was detected by western blotting. Pgk1 served as loading control. (G) Same assay as shown in (F) but using an anti‐Ape1 antiserum for western blotting. (H) Western blots of yeast cell lysates using an anti‐Atg8 antiserum. (I) Ape1 protease protection assay with the indicated yeast strains. The percentages below the blots indicate the percent of protected ApeI in the +protease K, −Triton X‐100 lanes and are the average of three independent experiments±the standard deviation. The numbers next to the blots indicate the molecular weight in kDa. Figure source data can be found with the Supplementary data.
HDAC6 is required for efficient lysosome–autophagosome fusion.Figure 2(A) Wild‐type and HDAC6 KO MEFs were transfected with pcDNA, pcDNA‐HDAC6WT, HDAC6CD, or HDAC6ΔBuz, along with mCherry‐GFP‐LC3 as indicated. Yellow signals indicate non‐acidic autophagosomes and red signals indicate acidic autophagolysosomes. Scale bar, 10 μm. (B) The total number of yellow vesicles was quantified from three independent experiments (>12 cells each) and presented as the percentage of total mCherry‐GFP‐LC3 dots (red plus yellow) along with the standard deviation. (C)In vitro fusion assays. Autophagosomes (APGs) and lysosomes (Lys) purified from wild type and HDAC6−/− MEFs were subjected to heterotypic and homotypic in vitro fusion assays (representative fields are shown in Supplementary Figure S3). Values are means+s.e. of the percentages of fusion from three independent experiments (more than 10 images per each experiment). (D) EM images of wild‐type and HDAC6 KO MEFs in normal growth conditions. Yellow arrows, autophagosomes; red arrows, autophagolysosomes; green arrowheads, multilamellar bodies. (E) Quantification of autophagosomes and autophagolysosomes. The error bar represents the standard error.
Figure 7A Joubert syndrome-associated INPP5E mutations (Bielas et al, 2009) used in this study. B N1E-115 cells stably expressing the indicated mutants treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf.A1 for 2 h, and analyzed by immunoblot using anti-LC3 and anti-GAPDH antibodies. The mutants were resistant to mouse siINPP5E because target sequence of the siRNA is different from the human correspondent sequence.C Quantitation of protein signal intensities from immunoblots in B showing differences in LC3-II levels in the presence and absence of Baf.A1 following normalization to the control proteinGAPDH. Results represent means ± s.d. of three independent experiments. **, P < 0.01; *, P < 0.05.
Double mutant screen for regulators of dynamic parameters Double mutant library, containing ~1600 strains with Hsp12-GFP reporter, was screened using automated flow-cytometry in six timepoints following exposure to stress (0.Figure 3Double mutant library, containing ~1600 strains with Hsp12-GFP reporter, was screened using automated flow-cytometry in six timepoints following exposure to stress (0.4M KCl). Estimated parameters (median over repeats) of all ~1600 double mutant strains. The color represents the total Hsp12-GFP produced. The same amount of GFP can be produced with multiple parameter combinations. The ellipses illustrate iso-expression areas in the graph. Three examples of individual strains (marked in B) with nearly the same total expression but dramatically different dynamics. Illustration of the genetic interaction trends of individual mutants. For each single mutant we plot the median of all the double mutants that contain it (purple dots). The gray dots are all individual double mutant strains (B). Illustration of the interactions of Δhog1 in the production rate parameter. Shown the production rate of the single mutant (X axis) versus the production rate of the double mutant with Δhog1 (Y axis). The gray line marks the Hog1 single mutant levels. Points close to the gray line are ones where the value of the double mutant is close to the value of Δhog1 and defined as epistasis of Δhog1 (pink dots). Illustration of the interactions of Δhog1 for the total open time parameter. Venn diagram comparing the number of epistatic pairs detected in various phenotypes.
Figure 8(A) Convertase-labeled bacteria were exposed to a concentration range of either preassembled C5b6 (C5b6MAC) or a mixture of C5 and C6 (Conv-MAC), in the presence of 100 nM C7. After washing, 20 nM C8 and 100 nM C9-Cy3 was added. Controls at 0 nM C5b6 or C5-C6 confirm that the detected C9-Cy3 deposition is specifically related to MAC formation (solid lines). Proper insertion of pores was assessed by a previously described shaving method with trypsin (Moskovich & Fishelson, 2007). Bacteria were first incubated with MAC components for 30 minutes and subsequently treated with 20 µg/ml trypsin for 15 min at 37°C (dotted lines). Data information: (A) Data represent mean ± SD of 3 independent experiments. (B) Convertase labeled perimCherry/cytoGFP bacteria (Green) exposed to C5b6MAC or Conv-MAC. Conditions were similar to those in (A), however C9-Cy5 was used to detect MAC pores (Red). 100 nM of C5 and C6 or C5b6 were used in combination with 100 nM C7, 20 nM C8 and 100 nM C9-Cy5. Conv + C5b6MAC and conv-MAC images were taken in separate experiments in which laser settings were adjusted to the staining intensity of C9-Cy5 to properly visualize pore distribution. Scale bars = 3 µm. (C Atomic force microscopy analysis of E. coli BL21 and MG1655 immobilized using the Poly-L-Lysine protocol. (C) Entire bacteria and high-resolution comparisons of untreated and convertase-labeled E. coli BL21 exposed to C5-C9 (Conv-MAC) for 10 minutes. Scale bars: 800 nm (left) and 30 nm (right). Height Scales: 1.01 µm (left), 8 nm (top right), 22 nm (bottom right). Width of magnification boxes (left): 42 nm, height scales: 8 nm (top left) and 13 nm (bottom left). Arrows highlight E. coli porin structures, an asterisk highlights the C5b-7 stalk. Height profiles (bottom) are shown for the white dashed lines in the images. (D) Atomic force microscopy (height and phase images) of convertase-labeled E. coli MG1655 exposed to a mixture of C5 and C6 (Conv-MAC) or preassembled C5b6 (Conv + C5b6MAC), in the presence of C7, C8, C9, FB and FD. Images were generated in the same experiment. Scale bars: 50 nm. Height scales: 15 nm.
Next Generation Sequencing reveals immunometabolic reprogramming in human T cells on KD.Figure 4A Schematic study flow: 44 healthy volunteers conducted a three week ketogenic diet (KD) with limited carbohydrate consumption of <30g per day. Blood was taken and analyzed before starting the diet (T0) and after three weeks of successful diet (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD8 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. B Total data set gene expression and differential regulation at the gene and pathway levels in human CD4+/CD8+ T cells prior to and post KD. Differential expression analysis was performed using DESeq2 while statistical significance was accepted for corrected p-values (FDR) smaller than 25%. C Heatmaps of z-scaled expressions of significantly differentially expressed genes in CD4+ T cells (left) and CD8+ T cells (right) prior to and post KD. Red color indicates an upregulation post KD, downregulated genes are indicated by blue color. D Volcano plots visualizing differential gene expression T1/T0 in human CD4+/CD8+ T cells. Log2 fold-changes (x-axis) and -Log10 FDR (false discovery rate) corrected p-values (y-axis) are shown for each gene. The dashed horizontal line depicts the FDR-threshold of 25%, significant genes in red. E Gene set enrichment analysis. Shown are the top ten up- and down regulated pathways ranked according to normalized enrichment score (NES).
Figure 1A. Schematic representation of domain organization of Mis18 proteins from S. pombe and human (as suggested by the Conserved Domain Database (CDD) and secondary structure predictions). B. Amino acid conservation of Mis18 proteins among eukaryotes. The alignment includes orthologues from S. pombe (sp), G. gallus (gg), M. musculus (mm), H. sapiens (hs) and B. Taurus (bt). Predicted (light orange; using PsiPred, http://bioinf.cs.ucl.ac.uk/psipred) and observed (bright orange; from the crystal structure shown in Fig 1D) secondary structure elements are shown below the aligned sequences. Amino acid residues mutated in this study are highlighted with circles (dimer interface residues) and asterisks (putative substrate-binding pocket residues). C. SDS-PAGE showing a representative fraction of the purified spMis18 N-terminal Yippee-like globular domain (amino acid residues 1-120; spMis181-120). D. Cartoon representation of the crystal structure of spMis181-120 in two different orientations, highlighting key residues within the putative substrate-binding pocket. E. Substrate-binding pocket mutations Y90A and Y74A/Y90A/T105A/S107K affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18-262 cells to varying degrees. Five-fold serial dilutions of mis18-262 cells transformed with plasmids harbouring the indicated spMis18fl constructs, spotted on PMG - uracil + Phloxine B media supplemented with (repressed) or without (expressed) thiamine, and incubated at the indicated temperatures; dead cells stain dark pink.