EMBO/sd-character-level-ner huggingface dataset:
Figure 2A-C qRT-PCR detection of mRNA levels of IL-1β, TNFα and IL-6 mRNA levels in CCDC50-WT and CCDC50-KO THP-1 cells primed with LPS and then treated with ATP and Nigericin (A), or infected with Listeria monocytogenes at a multiplicity of infection (MOI) of 10 and Salmonella typhimurium (MOI of 10) for 6 h (B) or MSU (40 μg/ml ) and SiO2 (40 μg/ml) for 6 h (C) (n = 3 biological replicates). Data information: L.m, Listeria monocytogene; S.t, Salmonella typhimurium; MSU, Monosodium urate; mRNA results are normalized to GAPDH and relative to untreated wild-type cells Data are representative of three biological replicates and shown as mean with SEM (A-C); **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test. D Enzyme-linked immunosorbent assays (ELISA) of IL-1β production in supernatants of THP-1 cells treated as in (A-C) (n = 2 biological replicates). Data information: L.m, Listeria monocytogene; S.t, Salmonella typhimurium; MSU, Monosodium urate; ; data are representative of two biological replicates (D). **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test. E-G CCDC50-WT and CCDC50-KO THP-1 cells were stimulated as in (A-C). The cells were collected for immunoblot analysis of pro-caspase-1, and the supernatants were subjected to cleaved caspase-1 and IL-1β analysis. The expression levels of cleaved-caspase-1 and cleaved-IL-1β were quantitated using ImageJ software. Data information: L.m, Listeria monocytogene; S.t, Salmonella typhimurium; MSU, Monosodium urate; Casp1, caspase-1; actin was used as a loading control.
Figure 6(A) Left panel, Autophagic flux, determined by the extent of lysosomal inhibitors-dependent increase in the ratio of lipidated to non-lipidated LC3 (LC3II/LC3I), is increased by 144DG11 (50µM, 24h). Right panel, 144DG11-mediated increase in autophagic flux, demonstrated by enhanced degradation of the autophagy substrate p62. Shown are representative immunoblots (grouped from different gels) and densitometric quantifications +/-s.d. (for LC3II/LC3I, n=4 biological replicates, *p<0.0023; for p62, n=5 biological replicates, *p<0.0099; two-tailed t-test). (B) Left panel, TEM images of liver tissue from vehicle or 144DG11-treated Gbeys/ys mice. High-magnification (right, scale bars=200 nm) and low-magnification (left, scale bars=1 µm) images show higher levels of glycogen/polyglucosan in lysosomes and cytosol, respectively. Right panel, quantification (+/- s.d) of lysosomal glycogen particles (n=3 biological replicates, *p<0.03, two-tailed t-test). G, Glycogen/polyglucosan; L, Lysosomes; M, Mitochondria. (C) 144DG11 reduces LC3 and p62 in mouse liver, but not muscle (n=3 biological replicates, *p<0.04 for LC3, **p<0.0007 for p62; two-tailed t-test, SEM). Scale bars, 10 µm (D) LAMP1-knockdown increases autophagic flux, which is further facilitated by 144DG11 (see densitometric quantification +/-SEM (n=3 biological replicates, *, p<0.1; **, p<0.05; ***, p<0.01 (two-tailed t-tests)). Immunoblots show LAMP1, LC3II/I, and actin in LAMP1 knocked-down and control APBD fibroblasts treated or not with 144DG11 and lysosomal inhibitors (LI). (E) LAMP1-KD and 144DG11 treatment cause lysosomal acidification. Upper panel, flow cytometry results showing that 144DG11 slightly increased acidification (yellow to blue median fluorescence ratio (Y/B)) in control, GFP-transduced, APBD fibroblasts (Y/B(GFP/144DG11)>Y/B(GFP), p<0.12), but significantly acidified LAMP1-KD, GFP-shLAMP1-transduced, APBD fibroblasts (Y/B(LAMP1-KD/144DG11)>(Y/B(LAMP1-KD), p<0.03). LAMP1-KD itself led to the most significant acidification (Y/B (LAMP1-KD)>Y/B(GFP), p<0.007). n=3, two-tailed t-tests. Middle panel Lysosensor staining of the corresponding cells. Yellow fluorescence intensity correlates with acidification. Lower panel, PAS (glycogen) staining of the corresponding cells. Scale bars, 50 µm. (F) ATP production rates (Figure 4B) +/- SEM in serum-starved (-S) and non-starved (+S) LAMP1-KD and GFP (Control) cells, untreated (UT), or treated for 24 h (CHR, chronic) or on assay (acute) with 144DG11. See text (144DG11 enhances LAMP1 knockdown-induced autolysosomal degradation and catabolism of glycogen, third paragraph) for results of statistical analysis (multiple t-tests, One Way ANOVA with Sidak's post-hoc corrections, n=3 biological replicates). (G) 144DG11 reduces lysosomal (LAMP1 positive) area in liver, but not muscle, of Gbeys/ys mice (n=3 biological replicates, *p<0.01, two-tailed t-test , SEM).Scale bars, 5 µm for liver (upper left panel), and 10 µm for muscle (lower left panel). Right panel shows quantification of the left panel.
Figure 7Global H3K27ac (A), Acly levels (B) in CD48- HSCs and CD48+ progenitor cells (HPCs) in the late phase after the administration of 5-FU. acetylated lysine (C), and ΔΨm (D) in CD48- HSCs and CD48+ progenitor cells (HPCs) in the late phase after the administration of 5-FU. (E) The enrichment of the gene set "HALLMARK_OXIDATIVE_PHOSPHORYLATION" within differentially expressed genes between CD48- HSCs and CD48+ progenitor cells. NES and q values represent normalized enrichment scores and FDR, respectively. (F) Average plot (top) and heatmap (bottom) of H3K27ac reads in CD48- HSCs and CD48+ HPCs in the late phase within cis-regulatory regions related to genes with lower (left) or higher (right) expression levels in CD48+ HPCs than in CD48- HSCs. (G) ATAC peaks of HSCs in the early or late phase, and H3K27ac peaks in CD48- HSCs or CD48+ HPCs in the late phase within the promoter and enhancer regions in the CD48 locus. (H) CD48 mRNA levels in CD48- HSCs and CD48+ HSCs in the late phase after the administration of 5-FU. (I) Average plot (top) and heatmap (bottom) of H3K27ac reads in CD48- HSCs and CD48+ HPCs in the late phase within accessibility-decreased (left: Green dot in Fig 3E), -unchanged (center: Blue dot in Fig 3E), or -increased cis-regulatory regions (right: brown dot in Fig 3E) in HSCs in the late phase. (J) Within accessibility-increased cis-regulatory regions in the late phase (brown dot in Fig 3E), 192 regions showed more than 2-fold increases in H3K27ac levels in CD48+ progenitor cells than in maintained HSCs (Venn diagrams). The GSEA plot represents the enrichment of the gene set related to these H3K27ac-increased regions within differentially expressed genes between CD48- HSCs and CD48+ progenitor cells. NES and q values represent normalized enrichment scores and FDR, respectively.
Persistent localisation of ATG16L1 to PAS inhibits autophagy.Figure 7ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LE-expression constructs and autophagy assessed during amino acid starvation (AA starve) followed by immunoblotting or immunofluorescence analyses. Immunofluorescence analyses of amino acid starved cells (2 hr) using antibodies against WIPI2 (red) or ATG16L1 (green). Scale bar: 9 μm. Cells as in (A) with the addition of 3'MA (and pretreatment with the drug for 30 min). Scale bar: 9 μm. Quantification of percentage of cells positive for ATG16L1 puncta Cells were left untreated, amino acid starved for 2 hr, or amino acid starved for 2 hr followed by refeeding with full growth media for 1 hr. Puncta formation was assessed by immunofluorescence staining using antibodies against S tag to detect ATG16L1.­­ Scale bar: 10 μm. Immunoblot analyses of amino acid starved cells in the presence of BafA1 for 2 hr. Cell lysates were analysed using the indicated antibodies. WIPI2-/- cells reconstituted with the indicated ATG16L1 constructs were amino acid starved for 2 hr. BafA1 treatment was included in all conditions for 2 hr prior to lysis. Cell lysates were analysed by immunoblotting using the indicated antibodies. Quantification of LC3-II/LC3-I ratio of three independent experiments is shown on the right panel. Ferroptosis assay in ATG16L1-/- cells stably expressing ATG16L1WT, ATG16L1LD or ATG16L1LE. Cells were cultured in amino acid free media (AA starve) in the presence of 10% FBS or 10% dialysed FBS (diFBS) for 24 hr. Quantification of percentage of PI-positive cells from at least three independent experiments is shown.
Figure 4Representative examples of single cell oscillators observed in CTRL (a) and MBSm (b) embryos imaged from 34hpf onwards; time series represent Her6::Venus expression relative to mKeima-H2B (top panel) and detrended relative signal (bottom panel); parameters reported for log-likelihood ratio (LLR) and period correspond to KOUosc . Pairwise analysis comparing proportion of oscillators in CTRL versus MBSm embryos indicated as dots; paired t test with two tail significance p<0.01**. Pairwise analysis comparing periods observed in CTRL versus MBSm oscillatory cells; dots indicate median per experiment per condition; paired t test with two tail not significant. Analysis of local coefficient of variation (LCOV) as a ratio between MBSm and CTRL; circles indicate median LCOV observed in MBSm cells divided by median LCOV observed in CTRL cells per experiment; bars indicate mean and SEM of LCOV ratios from 4 independent experiments. Representative examples of detrended Her6::Venus relative to mKeima-H2B in non-oscillatory single cells observed in CTRL (f) and MBSm (g) embryos imaged from 34hpf onwards. Aperiodic KOU covariance model indicating differences in rate of correlation decay between subsequent peaks corresponding to examples in (f-g). Example of one experiment representing quantification of noise by aperiodic lengthscale in one CTRL versus one MBSm embryo; bars indicate mean with 95% confidence intervals. Pairwise comparison of aperiodic lengthscale; dots indicate median per experiment from CTRL (15 cells, 5 cells, 13 cells, 7 cells; 4 embryos) and MBSm (14 cells, 5+12 cells, 4+8 cells, 13 cells; 6 embryos); paired t-test with two-tail significance for p<0.05 *.
Figure 2Cluster analysis of RNA expression of WT, cTAZ-/-, and cTAZ-/- /GFP-cTAZ put-back RKO cells. Genes down- and up-regulated more than 2 folds (P < 0.05) in cTAZ KO cells were included in cluster analysis. Green and red indicate down- and up-regulated genes respectively. Gene ontology (GO) analysis identified differentially regulated pathway by cTAZ. cTAZ suppressed expression of ISGs. RNA levels of selected ISGs in WT, cTAZ-/-, cTAZ-/-/GFP-cTAZ RKO cells were quantified by qPCR. Error bars indicate SD, n =3. **P<0.01; ***P<0.001; Student's t-test. cTAZ repressed 5×ISRE-luciferase activity induced by IFN-α. HEK293A cells were transfected with 5×ISRE-luciferase reporter along with the indicated plasmids, treated with or without IFN-α (50 ng/ml) for 12 hours. The expression of ectopic genes was determined by IB. Error bars indicate SD, n =3. ***P<0.001; one-way ANOVA test was used for statistical analysis. Knockout of cTAZ promoted ISGs expression in the presence of IFN-α. WT and cTAZ-/- RKO cells were treated with or without of IFN-α (50 ng/ml) for indicated time, the expression of selected genes was quantified by qPCR. WT or cTAZ-/- group was normalized by basal level, respectively. Error bars indicate SD, n =3. *P<0.05; **P<0.01; Student's t-test.
Figure 3Distribution of the telomere repeat array found at the junction between two fused chromosomes of senescent cells transduced with lentiviral vectors expressing either shControl or shRAP1. Variant telomere repeats were included in the estimation of the telomere array length. Data represent median +/- interquartile range (green lines) of 3 biological replicates. Statistical analysis was performed using Mann-Whitney U test ***p < 0.0001). Examples of DNA sequences found at the telomere fusion points of shControl and shRAP1 conditions. Telomere repeats are underlined in blue while variant telomere repeats are underlined in red. The sequence ID corresponds to the identifier in Table EV1.
Inflammation-mediated loss of mitochondrial integrity are reversed with genetic and pharmacological inhibition of iNOS.Figure 8Male C57BL/6 wildtype (WT) and iNOS knockout (KO) mice were intraperitoneally injected with 1mg kg-1 LPS or an equivalent volume of carrier solution. Control WT, Control KO, and LPS-treated KO cohorts were pair-fed (PF) to the WT LPS-treated cohorts. After 18h, mice were euthanized, and tibialis anterior muscles were imaged by transmission electron microscopy. (left) Representative micrograph of tibialis anterior muscle (n=2). Scale bar = 1 µm. (right) Zoomed section of representative image to highlight mitochondria. Scale bar = 0.5 µm. Male BALB/C mice were injected subcutaneously with C26 cells (1.25x106 cells) or an equivalent volume of saline. After 5 days, and everyday thereafter, saline and C26 injected mice were treated with or without GW (5mg kg-1). After 16 days, mice were euthanized and gastrocnemius muscles were imaged by transmission electron microscopy. (left) Representative micrograph of gastrocnemius muscle (n=2). Scale bar = 1 µm. (right) Zoomed section of representative image to highlight mitochondria mitochondria. Scale bar = 0.5 µm.
Assembly of RHIMs gives rise to homomeric and heteromeric structures.Figure 3Fluorescence polarisation profiles from incorporation of FITC-M451‑90 into excess unlabeled M451‑90 (green), RIPK1497‑583 (red), RIPK3387‑518 (blue) or ZBP1170-355 (purple). Average polarisation profile from smoothed triplicate samples plotted, with s.d. indicated. Experiment performed twice and representative results shown here Schematic representation of fluorescence spectroscopy experiments used to detect the formation of homo- and heteromeric amyloid fibrils. Fusion proteins containing RHIMs and different fluorescent partners are mixed together under assembly-permissive conditions and examined in the confocal volume. Co-assembly of two different fluorophores within one complex results in detection of coincident signals in the two channels Representative fluorescence intensity trace showing homomeric assembly over 40 s in mixtures of (C) mCHERRY-RIPK1497‑583 and YPet-RIPK1497‑583 and (D) mCHERRY-RIPK3387‑518 and YPet-RIPK3387‑518. E, F Representative fluorescence intensity traces showing heteromeric assembly over 40 s in mixtures of (E) mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 (inset is expansion of region indicated by *) and (F) mCHERRY-RIPK1497‑583-AAAA and YPet-RIPK3387‑518. All mixtures examined in at least three different experiments on three separate occasions
Figure 9(A) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22-23 WT mice before (Ctr WT) and after application of 2.5 µM DL-TBOA (TBOA 2.5) (n = 4; N=2) (right panel). The traces on the left are the corresponding average normalized STCs, isolated as in Figure 1C. The STC τdecay in TBOA 2.5 is 32 ± 2 % higher than in Ctr WT (paired t-test: P = 0.002). The left top panel shows the time course of τdecay of the transient component (due to the STC) of the current recorded in an astrocyte during a representative experiment in which TBOA 2.5 was applied at the time indicated by the horizontal bar. The steady-state effect was reached within 10-15 minutes from the beginning of the drug perfusion. (B) CSD threshold and velocity measured in cortical slices from P22-23 WT mice after perfusion for 20 minutes with extracellular solution without (Ctr WT: n = 23; N = 15) and with 2.5 µM DL-TBOA (TBOA 2.5: n = 25; N = 8). CSD threshold in TBOA 2.5 is 36 % lower than in Ctr WT (Mann-Withney U test test: P < 0.0001) and CSD velocity is 20% higher (unpaired t-test: P < 0.0001). (C) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22-23 WT mice before (Ctr WT) and after application of 1.5 µM DL-TBOA (TBOA 1.5) (n = 4; N=4). The STC τdecay in TBOA 1.5 is 22 ± 3 % higher than in Ctr WT (paired t-test: P = 0.004). (D) CSD threshold and velocity measured in cortical slices from P22-23 WT mice after perfusion for 20 minutes with extracellular solution without (Ctr WT: n = 23; N = 15) and with 1.5 µM DL-TBOA (TBOA 1.5: n = 18; N = 6). CSD threshold in TBOA 1.5 is 23 % lower than in Ctr WT (Mann-Withney U test test: P < 0.0001) and CSD velocity is 13 % higher (unpaired t-test: P = 0.003).
Increased insulin sensitivity in adipose-specific QKI-deficient mice.Figure 2Representative CT images of WT fl/fl and QKI KO fl/fl:AP2-Cre mice on an NCD or HFD, showing the whole-body fat composition. Glucose tolerance tests (GTT) and Insulin tolerance tests (ITT) were performed on WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. Blood glucose concentrations during GTT and ITT were monitored. (n= 10, per genotype) Blood glucose and plasma insulin levels in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (n = 8, per genotype) Plasma Free Fatty Acid (FFA) and Glycerol levels in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (n = 8, per genotype) H&E and Oil Red O staining, and liver triglyceride (TG) content in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (Scale bar represents 50 μm)
Figure 6(A) Schematic representation of a model for DISC structure consistent with the experimental observations in this paper. Key: green chevrons: FADD DED; orange chevrons: FLIP DED1/2; purple chevrons: caspase-8 DED1/2; black triangle: trimeric TRAIL-R2. (B) NanoBiT® assay performed on TRAIL-R2 DISC IPs (90') carried out in U20S Caspase-8 null cells co-transfected with wild-type or F114A FLIP-LgBiT fusion proteins and wild-type or F122A caspase-8-SmBiT fusion proteins. (C) Docking Connolly surface models of interactions between the DEDs of FADD (green), FLIP (orange) and caspase-8 (purple). (D) Potential DISC structure linking two receptor trimers via DED chains. Putative caspase domain interactions are depicted. (E) Western blot analysis of size exclusion chromatography experiments in which HCT116 cells transfected with Flag-tagged wild-type (WT) FLIP(S) were treated with 100ng/mL IZ-TRAIL for 3h prior to cell lysis and separation of low and high MW complexes using a size exclusion column. The fractions eluted at 2MDa and >2MDa are presented.
Figure 7Schematic diagram for the regulation of ETS-4 by TDPT-1 in axon regeneration.
Figure 5In the apo state of Sec complex, the Sec61 channel is closed by the plug while the lateral gate is already open. Sec62 flexibly associates with the Sec complex mainly through a basic surface on its Sec62 domain interacting with the acidic C-terminus of Sec63. During the insertion of a post-translational client, its signal sequence (ss) binds to the groove at lateral gate supported by the Sec62 TM2. At the same time, the Sec62 TM1 pushes the Sec61α TM3 outwards leading to an even more open lateral gate. This also leads to removal of the plug away from the pore ring, allowing, the translocating peptide to be gated through the Sec61α central pore.
Stability of the Bak and BclxL soluble domains.Figure 3(a) Comparison of the thermal stability of BclxLΔTM (left) and BakΔTM (right) measured by DSC (top) and Far-UV-CD at 222 nm (bottom). ) RDC-refined structure of BakΔTM. The NMR peak height ratio of the deuterated vs. protonated sample was used to identify solvent-protected amino acid residues (red spheres). ( (c) Comparison of the GuHCl-induced chemical unfolding of the secondary structure (black), monitored by far-UV-CD at 222 nm, and tertiary structure (green), monitored by Trp-fluorescence, of BclxLΔTM (left) and BakΔTM (right). The native (N), intermediate (I) and unfolded (U) states are indicated at the top. (d) Chemical shift perturbation (CSP) plot derived from 2D-[1H-15N] HSQC spectra of BakΔTM (top) and BclxLΔTM (bottom) +/- 1.5 M GuHCl. The most significant chemical shift perturbations (> mean value plus standard deviation, dotted line) are highlighted in red on the structures on the right.
a) MSCV retroviral constructs introduced in P14 cells (Supplementary Fig.figf2(a) MSCV retroviral constructs introduced in P14 cells (Supplementary Fig. 2a) to probe for autophagy activity, showing the transgenes encoding GFP-tagged wild-type LC3b (top; WT GFP-LC3b) and the GFP-tagged G120A mutant of LC3b (bottom; G120A GFP-LC3b). LTR, long terminal repeat; IRES, internal ribosomal entry site. (b) Flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on days 5, 8 and 30 after infection of recipients with LCMV. Numbers in blue outlined areas indicate percent GFP− cells among transduced (Thy-1.1+) P14 cells. (c) Summary of results in b, showing autophagy activity as percent GFP− cells. *P ≤ 0.0005 (unpaired t-test). (d) Multispectral-imaging flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on day 8 after infection, showing GFP signals from GFP+ transduced (CD45.1+ Thy-1.1+) P14 cells. Original magnification, ×60. (e) Summary of results in d, presented as the frequency of cells with more than one GFP+ punctum among vector transduced Thy-1.1+ P14 cells. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.). Data are representative of three independent experiments (b,d) or three independent experiments with three or more mice per group (e; error bars, s.e.m.) or six mice per group (c; error bars, s.e.m.).
increases the expression of gluconeogenic genes, inhibits insulin signaling and activates the mTOR pathway in the liver.Figure 3Eight-week-old male WT and DOCK5-/-mice were fed a SD or a HFD for 3 months. Mice were treated with insulin (1 U/kg) or control (PBS) by intraperitoneal injection for 10 minutes; animals were killed and livers were collected. The expression of expression of phosphoenolpyruvate carboxykinase(PEPCK) and glucose-6- phosphatase(G6Pase) mRNA and protein. Eight-week-old male WT and DOCK5-/-mice were fed a SD or a HFD for 3 months. Mice were treated with insulin (1 U/kg) or control (PBS) by intraperitoneal injection for 10 minutes; animals were killed and livers were collected. Total and phosphorylated insulin receptor (InsR), IRS-1ser1101 and Akt. Eight-week-old male WT and DOCK5-/-mice were fed a SD or a HFD for 3 months. Mice were treated with insulin (1 U/kg) or control (PBS) by intraperitoneal injection for 10 minutes; animals were killed and livers were collected. Total and phosphorylated mTOR, S6K1, and the levels of Raptor and Rictor protein.
Figure 3Unrooted Maximum-likelihood phylogenetic tree of 10 bacterial species used in this study based on multiple sequence alignment of 16S rRNA genes (distance scale of 0.1) using ClustalOmega. Transcriptional activities (Tx) of 421 regulatory sequences that were active in all 10 bacterial cell-free expression systems. Pairwise comparison of transcriptional profiles of 421 universally-active regulatory sequences between bacterial species. Example scatter plots with relatively high and low Pearson correlation in the heatmap (marked i and ii) are shown on left. Correlation between evolutionary divergences (16S rRNA percent identity) and pairwise Pearson correlation of transcriptional profiles. Dashed line represents linear regression. Activity profiles (Tx) of regulatory sequences from donor phyla Proteobacteria and Firmicutes in 10 bacterial species. GC contents of regulatory sequences from the phylogenetic groups are shown on right. Box plots are displaying the interquartile range (IQR) with median values (black line) and whiskers extending to the highest and lowest points within 1.5x the IQR. For normalization, transcription levels in log10 scale were transformed to Z-scores. All measurements are based on two biological replicates.