EMBO/sd-character-level-ner huggingface dataset:
Schematic illustration of how Aut2p and Aut7p are involved in the attachment of autophagosomes to microtubules allowing their directed movement to the vacuole.Figure 8Schematic illustration of how Aut2p and Aut7p are involved in the attachment of autophagosomes to microtubules allowing their directed movement to the vacuole. Aut2p interacts with Tub1p and Tub2p of microtubules.
SPRR5 is indispensable for proper keratinocyte differentiation A-C siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation (A) leads to decreased expression of key differentiation markers as shown by qRT-PCR (B) (FC = fold change) as well as western blot analysis (C) (n = 3-5 cultures/knockdown group).Figure 4siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation (A) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant). siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown by qRT-PCR (B) (FC = fold change) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant). siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown western blot analysis (C) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant). D SPRR5 knockdown efficiency in day 3 (D3) organotypic epidermal tissue (n = 4 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant). E SPRR5 depletion in regenerated organotypic epidermal tissue results in drastically reduced expression of differentiation proteins as shown by immunofluorescence analysis. Collagen VII (Col VII, green) stain indicates the basement membrane, nuclei are shown in blue and the differentiation proteins filaggrin and loricrin are shown in red (n = 4 tissue cultures/knockdown group; one exemplary picture for each group is depicted). Scale bar: 50 µm. F SPRR5 knockdown in regenerated epidermal tissue results in reduced transcript levels of key differentiation genes as obtained by qRT-PCR (n = 4 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).
Figure 4A. Correlation analysis process: miRNA and mRNA profiling datasets were cross-checked for miR-132 predicted targets that are significantly anti-correlated to miR-132 expression and significantly upregulated at late stages of AD. B. Positive hits were validated by semi-quantitative qPCR in ant-132-injected mousehippocampus. Black line indicates mean of scramble control set to 1. Dashed line indicates chosen threshold of 1.3-fold. Sample size, n=9 per group. Values were normalized to scramble-injected group and presented as mean ± SEM.
Figure 1(A) Schematic representation of the "Nucleation & caging" model. Most ParB dimers (green dots) are highly confined in a cluster (dotted circle) centered on the parS sites (black rectangles) onto which some ParB are stably bound (red dots). The DNA entering the cluster is bound stochastically by ParB. Red and blue lines represent DNA present at small and large (or on a different molecule) genomic distance from parS, re (B) ParB clusters on F plasmid in vivo. Typical E. coli cells (DLT3594) displays foci of ParBF-mVenus protein (top) expressed from the endogenous genetic locus of the F plasmid (F1-10B-mVenus). The nucleoid is labelled with Hu-mCherry (central). The overlay (bottom) combines the two fluorescent channels. Over 99 % of cells harbor ParBF foci. White bars: 1 µm (C) ParBF binding outside parSF on the F plasmid is compatible with a power-law decay. High resolution ChIP-seq performed on DLT3586 carrying the F plasmid (F1-10B). The ParB density, normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction, is displayed over 14-Kbp on the right side of parSF. Monte Carlo simulations and analytic formula are represented in red and dotted black lines, respectively. MC simulations were performed with a Freely-Jointed Chain of linear length L=15-Kbp and a cluster radius σ=75nm. The two other parameters, the Kuhn length a=10-bp and the total number of proteins on the F plasmid Nt=360 (related to the normalization constant of the protein concentration κ=0.41) were fitted from the ChIP-seq data (see text and Fig. EV2). As a benchmark for simulations, the analytics are obtained from Eq.(1) with the same parameters. Inset; The ParBF binding profile (black line) is represented as the number of nucleotide reads over 80-Kbp centered at parS. The number of reads in the Input sample (grey line) is normalized to the total number of reads in the IP sample Typical E. coli cell displays foc expressed from the endogenous genetic locus of the F plasmid (F1-10B-mVenus). The nucleoid is labelled with Hu-mCherry (central). The overlay (bottom) combines the two fluorescent channels parSF inserted at the xylE locus on E. coli chromosome from DLT358 ParBF binding outside parSF on the F plasmid is compatible with a power-law decay. High resolution ChIP-seq performed on DLT3586 carrying the F plasmid (F1-10B). The ParB density, normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction, is displayed over 14-Kbp on the right side of parSF. Monte Carlo simulations and analytic formula are represented in red and dotted black lines, respectively. MC simulations were performed with a Freely-Jointed Chain of linear length L=15-Kbp and a cluster radius σ=75nm. The two other parameters, the Kuhn length a=10-bp and the total number of proteins on the F plasmid Nt=360 (related to the normalization constant of the protein concentration κ=0.41) were fitted from the ChIP-seq dat parSF inserted at the xylE locus on E. coli chromosome fro DLT207
Infiltrating microglia phagocytose non-apoptotic photoreceptor rods during rod degeneration.Figure 3A-C Phagocytosis of rods by infiltrating microglia. (A) Representative example of a Iba1+microglial process extending into the ONL with a phagosome at its terminal end. Each phagosome contained a photoreceptornucleus (labeled with DAPI, arrow) that was identified as a rodphotoreceptor by rhodopsin immunopositivity (superposition of Iba1+phagosome with rhodopsin+soma in orthogonal views). (B) Example of an amoeboid microglia in the ONL with multiple phagosomes containing both rhodopsin-positive (arrowhead) and rhodopsin-negative (arrow) nuclei. (C) Rhodopsin+nuclei can be localized within CD68-positive phagosomes in infiltrating microglia, indicating phagocytic engulfment of rods. Scale bar, 10 μm.D, E The peak of microglial phagocytic activity occurred around P21 and declined subsequently, as measured by the number of rods phagocytosed (D) and the number of photoreceptors phagocytosed per microglial cell (E) (n = 3 animals at each time point). F-J Infiltrating microglia phagocytose TUNEL-negative rods in the ONL. (F) Although the peaks of TUNEL positivity and microglial phagocytosis occurred concurrently, the majority of rodphotoreceptors undergoing microglial phagocytosis were non-apoptotic (arrows), while many apoptotic photoreceptors were not phagocytosed by microglia (arrowheads); representative example of a P21 retina demonstrates non-overlapping patterns of TUNEL positivity (red) and microglial phagocytosis (Iba1, green). (G) Confocal analysis of a representative amoeboid microglial cell demonstrates that while the photoreceptornuclei in phagosomes were rhodopsin-positive (white), they were mostly TUNEL-negative. Only a small fraction of microglialphagosomes contained TUNEL+, rhodopsin+rods (example shown in H). Scale bars, 20 μm. (I) Quantitative analysis of photoreceptornuclei within microglialphagosomes according to rhodopsin (black symbols), and TUNEL labeling (red symbols) at different times during rod degeneration. (J) Quantitative analysis of all photoreceptors undergoing either apoptosis or phagocytosis at P21-23 demonstrates that TUNEL+ apoptotic photoreceptors and phagocytosed photoreceptors consist of two distinct populations, indicating separate but parallel mechanisms of rod degeneration. Quantitative analyses involved retinal sections from n = 3 animals at each age.
Figure 5A) Full-length TDP-43, mutant, and deletion fragments targeting regions that mediate self-assembly. Amino acid residue positions are indicated for each. B) Phase separation of full-length and TDP-43 variants (4 μM) mixed with A(GU)30 RNA (0.8 μM) or no RNA control at 250 mM NaCl observed by brightfield microscopy. Representative images for 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm.
Resident CD8+ T cells support chronic parenchymal inflammation and fibrosis in aged hosts.Figure 6.A. Experimental procedure of high or low dose of CD8 antibody (αCD8) treatment starting at day 21 post-infection. B. Lung-resident CD8+ T cell numbers in aged (left panel) or young (right panel) mice following CD8 Ab treatment. C, D. H&E staining of whole left-lung lobes from young (C) or aged mice (D) treated with indicated Abs. E. Percent of left lung lobes infiltrated by white blood cells via Image J analysis of D. F. Quantitation of resident monocytes (Ly6C Hi, ivCD45-CD64-CD11cLoSiglec-F- CD11bHiLy6CHi or Lo) and neutrophils (Neu, ivCD45-Ly6GHiCD11bHi) in IgG or high dose CD8 depleted young (left) or aged (right) mice treated with indicated Ab. G. Heat map for select DEGs of the lungs from aged mice treated with IgG (in triplicate), high dose (αCD8Hi, in triplicate) or low dose (αCD8Lo, pooled n=3) of CD8 Ab as analyzed by Nanostring. Orange is relatively high expression and purple is relatively low expression. H. Representative 200x micrographs of Mason's trichromatic stain on lung sections of young mice with or without high dose CD8 Ab treatment. I. Hydroxyproline levels reflective collagen content of the lungs from young mice with or without high dose CD8 Ab treatment. J. Representative 200x micrographs of Mason's trichromatic stain on lung sections of aged mice received control Ab, low dose of CD8 Ab (αCD8Lo) or high dose CD8 Ab (αCD8Hi) treatment. K. Hydroxyproline levels reflective collagen content of the lungs from aged mice received control Ab, low dose of CD8 Ab (αCD8Lo) or high dose CD8 Ab (αCD8Hi) treatment. B, F, H-K are 2 pooled experiments, E & G are single replicates. * p<0.05 Student's two-tailed t-test with unequal variance (I) or for ANOVA with correction for multiple tests (B, E, F, J, & K).
a) Anti-Atg8 western blot of the input and pellet fractions of a pulldown experiment using GST–Atg19 as bait and Atg8 as prey.figf2a) Anti-Atg8 western blot of the input and pellet fractions of a pulldown experiment using GST-Atg19 as bait and Atg8 as prey. The black vertical line indicates that unrelated samples were run on the same gel but in between the two samples on the left and the one on the right (Supplementary Fig. 7). (b) Coomassie stained gels of pulldown experiments using the GST-prApe1 propeptide as bait to pull down Atg19, the Atg19-Atg34 chimaera and Atg34, respectively. The Atg19-Atg34 chimaera contained residues 1-361 of Atg19 and residues 348-412 of Atg34. (c) Yeast two-hybrid (Y2H) assay testing for the interaction of Atg11 with Atg19, the Atg19-Atg34 chimaera and Atg34, respectively. (d) Anti-Atg8 western blot using the indicated GST fusion proteins as bait to pull down Atg8. The pulldowns were conducted in the presence or absence of the prApe1 propeptide. See Supplementary Fig. 1 for input gel. All experiments shown have been carried out twice. Images of uncropped western blots and gels can be found in Supplementary Fig. 7.
β‐Catenin deletion increases LC3 puncta and p62 expression in mouse intestinal epithelium in vivo.Figure 3(A-D) LC3 staining (red) in mouse intestinal crypt and villus epithelium increased (white arrowheads) 2 days post β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice (C and D) compared to control β‐catenin+/lox‐villin‐creERT2 mice (A and B). Further magnification of the areas marked by white squares is shown (lower panels). (E-H)p62 staining (green) increased in the crypt and villus epithelium2 days after β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice (G and H) compared to control in β‐catenin+/lox‐villin‐creERT2 mice (E and F). DAPI staining (blue) identifies nuclei. β‐Catenin deletion was confirmed by immunofluorescence ( Supplementary Figure S1B). Red and green channel levels were adjusted post acquisition for clarity (equal level changes applied across the entire figure). Further magnification of the areas marked by white squares is shown (lower panels). (I and J) Western blotting on extracts from mouse intestinal epithelial tissue demonstrated increased p62 and LC3‐II protein expression 2 (I) and 4 (J) days post β‐catenin deletion in β‐catenin−/lox‐villin‐creERT2 mice compared to control β‐catenin+/lox‐villin‐creERT2 mice.Source data for this figure is available on the online supplementary information page.
Coq9Q95X mice at 21 postnatal days and analysis of COQ9 protein.Figure 1Coq9Q95X mice at 21 postnatal days showing the loss of corporal hair. Representative Western blot images of COQ9protein in kidney homogenate from Coq9+/+ (n = 4) and Coq9Q95Xmice (n = 4) at 3 months of age. Antibody sc-271892 was used to map the C-terminal region of the COQ9protein and antibody ab-104189 was used to map the internal sequence of the COQ9protein. High-resolution LC-MS/MS proteomic analysis of kidneymitochondria from Coq9+/+ (n = 3) and Coq9Q95Xmice (n = 3) at 3 months of age. None of the six peptides of the COQ9 protein identified in Coq9+/+mice was detected in Coq9Q95Xmice.
IP3R1 silencing favors frequent changes in direction in immature DCs migrating in micro‐channelsAnalysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).Figure 3Analysis of shScramble (gray)‐, shIP3R(1,3) A (blue)‐, shIP3R(2,3) B (red)‐, and shIP3R(1,3) C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3) A and shIP3R(1,3) C and two independent experiments for shIP3R(2,3) B).A Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels. Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test. Analysis of shScramble (gray)‐, shIP3R(1,3) A (blue)‐, shIP3R(2,3) B (red)‐, and shIP3R(1,3) C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3) A and shIP3R(1,3) C and two independent experiments for shIP3R(2,3) B).B Dot plot showing velocity fluctuations and migration speeds of shScramble (gray)‐ and shIP3R(1,3) C (green)‐expressing DCs. Analysis of shScramble (gray)‐, shIP3R(1,3) A (blue)‐, shIP3R(2,3) B (red)‐, and shIP3R(1,3) C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3) A and shIP3R(1,3) C and two independent experiments for shIP3R(2,3) B).C Inverted sequential epifluorescence images (20×) of a representative control DC (left) and an immature shIP3R(1,3) C‐silenced DC (right) migrating in micro‐channels. DCs were differentiated from LifeAct‐GFP transgenic mice and imaged every 2.2 min. Analysis of shScramble (gray)‐, shIP3R(1,3) A (blue)‐, shIP3R(2,3) B (red)‐, and shIP3R(1,3) C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3) A and shIP3R(1,3) C and two independent experiments for shIP3R(2,3) B).D Percentage of DCs changing their direction of locomotion while migrating in micro‐channels. P‐values were calculated with a paired test with respect to the shScramble condition. Analysis of shScramble (gray)‐, shIP3R(1,3) A (blue)‐, shIP3R(2,3) B (red)‐, and shIP3R(1,3) C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3) A and shIP3R(1,3) C and two independent experiments for shIP3R(2,3) B).E Dot plot showing the frequency of changes in direction of immature DCs while migrating in micro‐channels. Bars show the mean plus the standard deviation. A Kruskal-Wallis test was applied for statistical analysis.
Figure 6A Representative western blot of primary hepatocytes starved in HBSS for the indicated time points and probed with indicated antibodies. Pan-actin was used as loading control. B, C Quantification of western blots as shown in (A). Antibody signal was normalized to pan-actin signal and signal of phospho-protein was normalized to signal of total protein (n=4 independent experiments). Data information: In (B, C), data shows the mean ± SEM. **, P≤0.01 for genotype and ***, P≤0.001 for time (Two-way ANOVA). Error bars show minimum and maximum values. D Representative western blot of total protein extracts from livers of starved Li-CluhWT and Li-CluhKO mice probed with indicated antibodies. GAPDH was used as loading control. E, F Quantification of western blots as shown in (D). Antibody signal was normalized to GAPDH signal and signal of phospho-protein was normalized to signal of the total protein (n=8 mice per genotype). Data information: data are presented as histograms showing the mean ± SEM. Error bars show minimum and maximum values. ***, P≤0.001 (Student's t-test). (H, J) *, P≤0.05; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test). G Confocal images of primary hepatocytes grown in HBSS for 16 h and stained with anti-cleaved caspase 3 antibody. Scale bar, 25 µm. H Quantification of cleaved caspase 3 positive hepatocytes treated with or without rapamycin. More than 100 cells per replicate were analyzed in a blind fashion (n=3 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. Error bars show minimum and maximum values. I IHC staining of cleaved caspase 3 in liver sections of starved Li-CluhWT and Li-CluhKO mice. Arrows point to positive apoptotic foci. Scale bar, 200 µm. J Quantification of positive apoptotic foci The number of positive spots was relativized to the area analyzed (n=5 mice per genotype). Data information: data are presented as histograms showing the mean ± SEM. K. Confocal images of primary hepatocytes grown in indicated media and stained with anti-G3BP1 and anti-mTOR. Scale bar, 10 µm. L Manders' colocalization coefficient between G3BP1 and mTOR staining (n >50 cells isolated from 3 mice). Data information: In (L), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001 (Student's t-test). (H, J) *, P≤0.05; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).
HIV induces autophagy in human macrophages.Fig 1(A) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (B) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h in the presence of pepstatin A, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3. (C) Macrophages were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated iodixanol velocity gradient purified HIVBa-L for 24 h. Cells were then harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.
Loss of USP42 leads to Wnt-dependent proliferation and EMT A, Scheme showing the proposed functions of ZNRF3/RNF43 and USP42 in colorectal cancer cells.Figure 5A, Scheme showing the proposed functions of ZNRF3/RNF43 and USP42 in colorectal cancer cells. Auto- and paracrine Wnt secretion is promoted by Evi (WLS) and PORCN. B,C, Volcano plots representing the genes that conform the Intestinal Stem Cell (ISC) signature (Merlos-Suarez et al., 2011) (B, n = 45 genes, Dataset EV1F), and the EMT signature in cancer cells (Rokavec et al., 2017) (C, n = 171 genes, Dataset EV1G) upon USP42 knockdown in HCT116 cells. Significance was capped at -Log10P = 24. D, Bright field images of HCT116 cells upon USP42 knockdown. Scale bar = 500 μm. E-H, Immunofluorescence microscopy showing endogenous E-cadherin levels in wt and WLS (Evi) KO HCT116 cells upon transfection with siUSP42 (E), or electroporation with eSpCas9 empty plasmid (non-edited) or eSpCas9-USP42 gRNAs(G). Scale bar = 20 μm. In (F,G), quantification of the fluorescent signal (Alexa-488) in n ≥ 4 biological replicates with n ≥ 15 cells per replicate are shown. I-L, Spheroid formation experiments with HCT116 cells upon transfection with siUSP42 (I-K) or electroporation with eSpCas9 empty plasmid (non-edited) or eSpCas9-USP42 gRNAs (L). Scale bar = 500 μm. Where indicated, cells were treated with 5 μM IWP-2 or 20 μM LGK-974 (PORCN inhibitors). The median size was quantified for n ≥ 13 spheroids from a representative experiment repeated independently n ≥ 3 is shown.
Figure 2A. Summary of protein-nucleic acid interactions. Solid and dashed lines represent polar interactions and hydrogen bonds, respectively. The −35 and −10 elements are colored in red letters. The extended −10 motif is in magenta. B. Recognition of the promoter −10 element. The RNAP is shown in surface representation: β subunit, cyan; β′ subunit, pink; β′ lid, green; β′ rubber, yellow; and σ28, wheat. The right and left side insets at the bottom part are the zoom-in views of the interactions on the NT-strand and T-strand, respectively. C. Conserved sequences around the −10 element of flagellar promoters (from −16 to −7 positions) generated by WebLogo Crooks et al, 2004(). D. Recognition of the promoter extended −10 element. E. Recognition of the promoter −35 element by the σ4 domain. F. Influence of mutating σ4 on promoter recognition.