EMBO/sd-character-level-ner huggingface dataset:
In carbon starvation, nucleosides and PPP compounds are produced by RNA degradation via autophagy in response to kinase signals.Figure 3(A) Experimental design for demonstrating metabolite production via macromolecule degradation. Yeast cells were grown on 2% unlabeled glucose, and then switched to U‐13C‐glucose for 70 min, which completely labels free metabolite but only partially labels macromolecules. Thereafter, glucose was removed and the metabolome analyzed by LC‐MS. (B) Fraction of unlabeled nucleosides, nucleic bases and PPP intermediates as a function of starvation time, in wild‐type and autophagy deficient (atg7 deletion) yeast. The x axis represents minutes after carbon starvation, and the y axis represents fraction of unlabeled metabolites (mean±range of N=2 biological replicates). (C) Ratio of metabolite levels in atg7 strain versus wild‐type strain in carbon starvation. (D) Ratio of metabolite levels in bcy1 strain and snf1 strain versus wild‐type strain in carbon starvation. (E) Ratio of metabolite levels in rapamycin treatment versus nitrogen starvation for wild‐type yeast. In (C) to (E), all reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.
Figure 2A, Multi sequence alignment of TFEB orthologs showing conserved homology between different species of the region containing TFEB Cysteine 212. B, Sequence homology analysis between human TFEB and TFE3 depicting the region containing the single cysteine residue in the entire proteins. Multi sequence alignment of TFE3 orthologs showing conserved homology between different species of the region containing TFE3 Cysteine 322. C, Immunoblot analysis of protein lysates from ARPE-19 cells expressing TFEB-FLAG-WT or mutants treated with 250 μM NaAsO2 or EBSS for 1h and run under non-reducing condition. D, Immunoblot analysis of protein lysates from ARPE-19 cells expressing TFE3-GFP-WT or mutants run under non-reducing condition. E, Immunoblot analysis of co-immunoprecipitated TFEB-FLAG and 14-3-3 from ARPE-19 cells over-expressing TFEB-WT or TFEB-C212S mutant. F, Immunoblot analysis of co-immunoprecipitated TFEB-FLAG, 14-3-3 and endogenous TFE3 from ARPE-19 cells over-expressing TFEB-WT or TFEB-C212A mutant. Immunoblots are representative of at least three independent experiments.
Figure 6(A and B) Summary of candidate secretory autophagy cargos, identified by quantitative proteomics based on isobaric tandem mass tags (listed in Table EV1) and sorted by cellular compartments in (A) or presence/absence of leader signal peptide in (B). (C) Ferritin levels in supernatants of THP-1 cells subjected to knockdowns as indicated and sequentially treated with LPS and LLOMe. (D) Ferritin levels in supernatants of THP-1 cells subjected to knockdowns as indicated and infected with M. tuberculosis as indicated. (E) Levels of ferritin were determine in supernatants from THP-1 cells that were subjected to knockdown as indicated and were sequentially treated with LPS and LLOMe. (F) Confocal microscopy of HeLa cells expressing GFP-Sec22b were treated with LLOMe, and stained for FTH1. Line tracings correspond to arrows. Scale bars, 5 μm;
Figure 6A Structure of the TRPML1-GCaMP6m-based Ca2+ sensor. B Colocalization of TRPML1-GCaMP6m (green) with LysoTracker (red) in Hela cells. Scale bar, 5 µm. C ML-SA1 (20 µM)-induced peak GCaMP6m responses (ΔF/F0) were increased in CRISPR-Cas9-mediated LAMTOR1 KD cells. N = 9-10 cells from 4 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Student's t-test *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, n.s., not significant. D TAT-2031 treatment (10 µM) increased ML-SA1-induced TRPML1 Ca2+ release in both control and LAMTOR1 KD cells. Data information: traces represent the mean values of each group. E Quantification of peak responses as shown in D. N = 11-19 cells from 5 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post hoc analysis *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, n.s., not significant. F Colocalization of TRPML1-GCaMP6m (green) with LysoTracker (red) in hippocampal neurons. Scale bar, 10 µm. G Treatment with GPN (200 µM), BAPTA-AM (20 µM), or ML-SI1 (20 µM) blocked ML-SA1-induced TRPML1-GCaMP6m responses in neurons. Data information: traces represent the mean values of each group. H Quantification of responses in G. N = 5-8 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, n.s., not significant. I Both LAMTOR1 KD and TAT-2031 treatment (10 µM) increased ML-SA1-induced TRPML1-GCaMP6m responses in neurons. J Quantification of peak responses as shown in I. N = 6-13 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post hoc analysis *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, n.s., not significant. traces represent the mean values of each group. K LAMTOR1 KD increased PI(3,5) P2 (0.5 µM)-induced TRPML1-GCaMP6m responses and the blocking effect of ML-SI1 treatment (20 µM). L Quantification of peak TRPML1-GCaMP6m responses as shown in K. N = 6-16 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post hoc analysis *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, n.s., not significant. traces represent the mean values of each group.
a,b) Immunofluorescence microscopy of HeLa cells expressing Flag–ΔN85-ATG16L1 (green; a) or Myc–FL-ATG16L1 (green; b) and HA-tagged Nod2 L1007fsinsC (HA–Nod2, 1007fs).figf7(a,b) Immunofluorescence microscopy of HeLa cells expressing Flag-ΔN85-ATG16L1 (green; a) or Myc-FL-ATG16L1 (green; b) and HA-tagged Nod2 L1007fsinsC (HA-Nod2, 1007fs). Area outlined at left is enlarged in other images. (c,d) Immunofluorescence microscopy of HeLa cells expressing Flag-ΔN85-ATG16L1 (c) or Myc-FL-ATG16L1 (d) together with HA-tagged Nod2fs (red). Blue, DAPI. Scale bar, 8 μm. (c,d) Immunofluorescence microscopy of HeLa cells expressing Flag-ΔN85-ATG16L1 (c) or Myc-FL-ATG16L1 (d) together with HA-tagged Nod2fs (red). Blue, DAPI. Scale bar, 8 μm. (e) Immunofluorescence microscopy of wild-type and Nod2fs-KI BMDMs transduced with GFP-LC3 (green) and infected for 0.5 h with S. flexneri M90T-RFP (red), followed by incubation for 1 h with gentamicin. Arrows indicate no colocalization of Nod2 with ΔN85-ATG16L1 or FL-ATG16L1. Scale bar, 15 μm. (f) Quantification of the colocalization of GFP-LC3 signals with S. flexneri M90T-RFP in experiments as described in e. Data are representative of one of three independent experiments (error bars (f), s.d.).
PINK1 accumulates in the mitochondrial matrix upon knockdown of Lon.Figure 4PINK1 accumulates in the mitochondrialmatrix upon knockdown of Lon.Immunofluorescent staining of thoracic muscles expressing matrix-targeted YFP (Mito-YFP) under control of the sqh genomic promoter, together with either (A) Myc-tagged MitochondrialRho (Miro-Myc), (B) FLAG-tagged Optic atrophy 1 (Opa1-FLAG), (C) endogenous Cytochrome C (Cyto C), (D) Myc-tagged PINK1 (PINK1-Myc), or (E) Lon-RNAi1 (Lon-R1) and PINK1-Myc. The muscle-specific Mhc-GAL4 driver was used to drive expression of Miro-Myc in panel A and Opa1-FLAG in panel B, and the muscle-specific Dmef2-GAL4 driver was used to drive expression of Lon-R1 in panel E. (F) We determined the percent volume shared between green objects (representing the matrix marker Mito-YFP) and red objects (representing the mitochondrial proteins analyzed in A-E). Red objects that shared at least 60% of their volume with Mito-YFP were considered to co-localize. Bar represents 2 µm.
Figure 4(A) Western blot analysis of protein in whole cell lysates (WCL) from BMDMs stimulated with LPS for 3 h and then treated with LicoB for 1 h (LicoB after LPS), or BMDMs first treated with LicoB for 1 h and then stimulated with LPS for 3 h (LicoB before LPS). (B) TNF-α production in the supernatant (SN) was measured using ELISA in the indicated samples in (A). (C, D) Quantification of intracellular K+ (C) and measurement of caspase-1 activity in the SN (D) of LPS-primed BMDMs pre-treated with the indicated dose of LicoB for 1 h and then stimulated with nigericin for 45 min. (E) The FLIPR Tetra® system was used to determine the effect of LicoB on ATP-induced Ca2+ flux in LPS-primed BMDMs. (F, G) Percentage of mtROS-producing cells in LPS-primed BMDMs pre-treated with LicoB for 1 h and then stimulated with nigericin 45 min, followed by staining with MitoSOX™ (G). (H) Caspase-1 activity measurement in the supernatant (SN) of samples described in (F). (I) Cell lysates of LPS-primed BMDMs treated with/without nigericin were incubated with sepharose or LicoB-sepharose. The pull-down samples and input were analysed using Western blot analysis. (J) Cell lysates of LPS-primed BMDMs were incubated with sepharose or LicoB-sepharose in the presence of different concentrations of free LicoB (0.5 mM and 1 mM). The pull-down samples and input were analysed using Western blot. (K) HEK-293T cells were transfected with Flag-NLRP3 or Flag-vector and then treated with LicoB (40 μM). Immunoprecipitation was performed with anti-DYKDDDDK (Flag) affinity gel agarose beads; Western blot analysis has been shown.
Figure 5Number of unique CAGE-supported TSSs mapping to a 10 nucleotide region upstream of motif-dependent 21U-RNAs. A clear enrichment of TSS 2 nt upstream of 21U-RNAs is observed. Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif-dependent piRNAs upon ints-11 RNAi knockdown in an N2 and a tfiis mutant background calculated from two merged replicates. N2 empty vector was used as a baseline for fold change calculation. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range. Heatmap showing changes in CAGE signal upstream of motif-dependent 21U-RNAs upon ints-11 knockdown in an N2 and a tfiis mutant background. The total cpms corresponding to each nucleotide position upstream to 21U-RNAs are shown as a barplot (top part). Length distributions of CAGE fragments initiating 2 nt upstream of motif-dependent 21U-RNAs. The total counts in each length bin are normalized to counts per million of mapped reads. Average normalized CAGE signal (transcripts per million) 2 nt upstream of motif-dependent and motif-independent piRNAs (excluding undetected loci). Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif-independent piRNAs upon ints-11 RNAi knockdown in an N2 and a tfiis mutant background. N2 empty vector was used as a baseline for fold change calculation. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range
Figure 3Representative fused PET/MRI images of the transverse view of the thoracic region of lean (n = 5), diet induced obese (DIO) (n = 4) and leptin deficient (ob/ob) (n = 5) mice that received the PDE10A radioligand [18F]-AQ28A. Interscapular brown adipose tissue (BAT) in each image is highlighted by the crosshairs. The mean standardized uptake (SUV) value of [18F]-AQ28A in BAT throughout the dynamic scan was calculated with associated area under the curve (AUC). * P = 0.0128 (lean vs DIO) and ** P = 0.0031 (lean vs ob/ob) using unpaired 2-tailed Student's t test.
Figure 5 [A] Clustering of DA-regulated immunity-related genes in Arabidopsis with sufficient Pi, as revealed by RNAseq analysis.  [B] DA suppresses plant ROS burst induced by flg22 or elf18. ROS accumulation was represented in relative luminescence units (RLU). Data bar indicate mean ± SE (n=24) readings for 60 min. Three independent experiments were performed and similar results were observed. **p < 0.01, student t-test.  [C] DA induces gene expression of AZI1 in Arabidopsis. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group. Bar indicates mean ± SE (n=3), two biological replicates were analyzed with similar results.  [D] flg22-induced MPK3/MPK6 phosphorylation in Arabidopsis is not decreased by DA. Kinase assays were performed at 5 min after flg22 treatment. Three independent experiments were performed and similar results were observed.  [E] Plant resistance to the pathogen Pst DC3000 is not reduced by DA. The severity of pathogen infection was measured as colony forming units (CFUs) per mg FW plants at 0 and 3 days after inoculation. Data bar indicate mean ± SE (n=8), two independent experiments were performed. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group.  [F] Root colonization of GB03 in wild type Arabidopsis (Col-0) and the mutants/transgenic lines npr1, NahG, and rbohd. *** p < 0.001compared with Col-0 without DA treatment, n= 9 biological replicates, student t-test.  [G] Bacteria chemotaxis with different concentrations of DA. Data point indicates mean± SE (n=6). Two independent experiments were performed and similar results were observed. *** p < 0.001, student t-test.
N-specific CTLs induced by anti-N antibodies and TRIM21 drive potent in vivo cell killing.Figure 4A) Splenocytes from uninfected CD45.1 mice, either pulsed with 3 concentrations of N peptide and cell trace violet (CTV) or unlabelled control cells, were transfused intravenously into WT and KO mice (CD45.2) that had been infected with 0.5 x 10^5 FFU LCMV 8 days earlier. After 3 hours, spleens from recipient mice were harvested and the proportion of CTV-labelled CD45.1 cells was analysed by flow cytometry. Histograms from single representative uninfected (UI), WT and KO mice are presented, showing the proportion of CD45.1 cells remaining for each of the labelled fractions normalised to mode. Summary data from all individual mice in the same experiment is presented in associated scatter plot, showing the mean ± standard error. B) Labelled splenocytes as for (A) were transfused into WT and KO mice that had been infected with LCMV 8 days earlier, and received mAb KL53 on days 1 and 3pi. Flow cytometry histograms from single representative mice of each genotype. Summary data from all mice in the experiment is presented, showing the mean ± standard error.
Increased E-cadherin:β-catenin levels in colonic crypts of wild-type mice.Figure 3Staining of small intestinal and colonic crypts for E-cadherin. Scale bar, 100 μm. Western blot of purified crypts from the small intestine and colon (N = 4 mice). qRT-PCR of whole tissue from small intestine and colon. Expression of mRNA (2(−dCt)) calculated relative to GAPDH (N = 3 mice). Statistics: one-sided Mann-Whitney U-test. Proximity ligation assay for E-cadherin and β-catenin. For each mouse (N = 6), at least 10 crypts per tissue were analysed to calculate the mean. Statistics: one-sided Mann-Whitney U-test; white scale bar, 50 μm.
Figure 3Vero cells were infected at MOI 1 (or mock infected) and treated with proteasome inhibitors MG132 (MG 20 µM), lactacystin (LC 20 µM), chloroquine (CQ 50 µM), 3-MA (5 mM), or bafilomycin-A1 (BAF-A1 0.2 µM) for the final 6h. Representative flow cytometry histogram of BTV-VP7 expression in inhibitor-treated cells. Vero cells were infected at MOI 1 (or mock infected) and treated with proteasome inhibitors MG132 (MG 20 µM), lactacystin (LC 20 µM), chloroquine (CQ 50 µM), 3-MA (5 mM), for the final 6h. Immunoblots probed for STAT1, STAT2, LC3 and GAPDH expression. Vero cells were infected at MOI 1 (or mock infected) and treated with bafilomycin-A1 (BAF-A1 0.2 µM) for the final 6h. Immunoblots probed for STAT1, STAT2, LC3 and GAPDH expression. Immunofluorescence of BTV-8 or mock-infected Vero cells (treated for the last 6h with BAF-A1) stained for BTV proteins, STAT2 and p62. Inset shows detail of STAT2 and p62 colocalization. Nuclei were counterstained with DAPI. Arrowheads in inset indicate p62 and STAT2 colocalization. Scale bar = 20 μm. Inset scale bar = 3 μm.
Figure 2(A) A representative FRET trace (blue line) was obtained as in Fig 1, and fit with a hidden Markov model (black dashed line). (B) Traces as in (A) obtained in the presence of different nucleotides, were used to determine the number of states best fit by the Markov model. (C) Transition density plot of idealized ATP FRET states obtained in (B). (D) The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1500 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. (E) As in (D), but with high FRET (1656 high FRET states). (F) Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (G) Distribution of FRET values determined from 168 traces as in (D) fit with a Gaussian model (black curve).
Figure 3A. Human orthologs of genes that were up- and down-regulated in Hnf1aaKO pancreas were also up- and down-regulated in human pancreas with low vs. high HNF1A expression (lowest vs. highest expression deciles, respectively). A random list of 717 genes controlled for similar expression levels was used for comparison. Violin plots include median and interquartile ranges. Dots are average values for each gene. Kruskall Wallis P<0.0001. B. GSEA demonstrates that down- or upregulated genes in Hnf1aaKO mice (downward or upward arrows) showed down- or upregulation, respectively, in gene lists ranked by differential expression in non-classical vs. classical PDAC molecular subtypes from the TCGA-PAAD study (Cancer Genome Atlas Research Network. Electronic address & Cancer Genome Atlas Research, 2017). All enrichments had GSEA FDR q values < 0.01. C. Analysis of HNF1A function in 121 high-purity cases of the ICGC-PACA-AU cohort identified tumors with most pronounced downregulation of direct HNF1A target genes. We performed GSEA with a gene set of 106 human orthologs of HNF1A direct targets showing downregulation in Hnf1aaKO pancreas. For each tumor sample, we performed differential expression against all other samples, and used GSEA to ascertain abnormal expression of the mouse HNF1A-dependent gene set in the tumor. Samples were ranked by the resulting normalized enrichment score (NES), and classified as either HNF1A LoF samples (purple, NES < 0; P < 0.05), or Control 1 (beige, NES < 0; P > 0.05) and Control 2 (grey, NES>0). HNF1A LoF samples were predominantly non-classical tumors (Collisson et al., 2011, Moffitt et al., 2015, Bailey et al., 2016). Putative loss of function KDM6A mutations (KDM6A LoF mutants) were found in 19% of HNF1A LoF tumors vs. 2% of all others (Fisher's P = 0.005). KDM6A mutations were considered functional if classified as ´high´ functional impact in ICGC (small ≤200bp deletions/insertions, single base substitutions), or as likely loss of function structural variants in (Bailey et al., 2016), all of which were frame-shift mutations. Other KDM6A mutations were classified as unknown. Heatmaps show Z-score normalized expression of deregulated genes in Hnf1aaKO pancreas. We confirmed that 85% of 106 downregulated and 60% of genes of 146 upregulated human orthologs showed differential expression across the 3 HNF1A profiles (q<0.05, SAM multiclass analysis). D. HNF1A mRNA levels differed in HNF1A LoF and control groups (Kruskal-Wallis, P<0.01), despite considerable variability and overlap between groups. E. KDM6A mRNA levels were downregulated in HNF1A LoF tumors (Kruskal-Wallis, P<0.001).Data information: Box plots in (D and E) show HNF1A and KDM6A expression in HNF1A LoF tumors (n=26) and Control 1 (n=39) and Control 2 (n=57) tumors. The horizontal central line marks the median. Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1.5 x interquartile range outside box limits.
Figure 4Quantification of the relative frequency of myofibre cross-sectional areas (CSAs) expressed as a frequency distribution in the TA and VM muscles of untreated mdx mice or mdx mice treated with a low or high dose of anti-IGF2R for 4 and 9 weeks (a). (b) Quantification of regenerating myofibres as a proportion of centrally nucleated fibres in the total number of fibres after anti-IGF2R administration. The quantification of regenerating and M-cadherin-positive satellite cells per section is shown. One-way ANOVA test, **p<0.01; ***p<0.001; ****p<0.0001. (c) Representative WB analysis of neonatal myosin heavy chain (neonatal MHC) expression and quantification expressed as the ratio of neonatal MHC/β-actin in untreated mdx mice and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; ***p<0.0001 (n=10 mice per group). (d) Specific force (maximal tetanic force normalized to cross-sectional area, Po/CSA) from TA and VM muscles in C57Bl6/J mice, untreated mdx mice, and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001 (n=10 mice per group). SERCA activity quantified as ATPase hydrolysis activity vs. min (e) in C57Bl6/J mice, untreated mdx mice, and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001 (n=10 mice per group).
Figure 3A. Anti-V5immunoprecipitates (IP) from HeLa cells expressing nothing (-) or V5-DKC1 were analyzed by immunoblotting for V5-DKC1 and qRT-PCR for the bound 7SK RNA. The error bars represent mean ± SD from three independent experiments. B. Anti-FlagIP from NE of HeLa or the HeLa-derived F1C2 cells expressing CDK9-F were analyzed by Western blotting (WB) for the indicated proteins and primer extension (PEx) for 7SK RNA. C. WB analysis of DKC1 and CDK9 levels in two HeLa clones inducibly expressing shDKC1 upon doxycycline (DOX) treatment for 5 days. D. The 7SK RNA levels in these two clones were detected by qRT-PCR before and after the DOX treatment and normalized to the GAPDH mRNA levels. The error bars represent mean ± SD from three independent experiments. E. 1D-TLC analysis of Ψ250 in 7SK RNA from inducible DKC1 KD clone 1 before and after the DOX treatment. F. NE and anti-CDK9IP from NE of inducible DKC1 KD clone 1, which was untreated or treated with DOX, were analyzed by WB for the indicated proteins and PEx for 7SK RNA. G. Clone 1 was treated with DOX for 3 days and then transfected with the Tat-Flag cDNA (20 ug/150-mm dish). Two days later, NE and anti-FlagIP from NE were analyzed as in F.