EMBO/sd-character-level-ner huggingface dataset:
Figure 8(A) Import of Mcp3 is mediated by the MIM complex. Mitochondria isolated from WT, mim1Δ, or mim2Δ cells were incubated with radiolabelled Mcp3 for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. Bands corresponding to the mature (m) form were quantified. Import after 15 min into wild-type mitochondria was set to 100%. The mean with standard deviations is depicted (n=3; SD). I, 20% of radiolabelled precursor protein used in each import reaction B) Steady state levels of over-expressed HA-Mcp3 are reduced in cells lacking MIM subunits. Crude mitochondria were obtained from WT cells containing an empty plasmid (Ø) and from wild-type, mim1Δ or mim2Δ cells containing a plasmid encoded HA-Mcp3. Samples were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, Tom20 as MIM substrate and Fis1 as a loading control. HA-Mcp3 levels were quantified in relation to Fis1 levels. Levels in wild-type cells were set to 100%. The bar diagram shows the mean with standard deviation (n=3; SD; *, p < 0.05; **, p < 0.01). (C) Mcp3 precursor binds in vitro to Mim1. Radiolabelled Mcp3 was incubated with amylose beads harbouring recombinant MBP or MBP fused to Mim1. After washing proteins were eluted with sample buffer. Analysis was performed by SDS-PAGE, blotting and autoradiography. To demonstrate equal amounts of MBP fusion proteins the membrane was stained with Ponceau S. I, input 5% of precursor used; n.b., 5% of unbound material;.b, 100% of bound material.
a) The N-terminal C2 domain or the C-terminal region of UVRAG interacts individually with Vps16.figf2(a) The N-terminal C2 domain or the C-terminal region of UVRAG interacts individually with Vps16. 293T cells were co-transfected with HA-Vps16, together with vector, Flag-UVRAG or Flag-UVRAG mutants. WCLs were immunoprecipitated with anti-Flag followed by immunoblotting with an anti-HA antibody. (b) HeLa cells were transfected with EGFP-Vps16 together with Flag-UVRAG mutants as indicated and stained with anti-Flag (red), followed by confocal microscopy. Scale bars, 5 μm. (c) Interactions of the UVRAG C-terminal truncated mutants with Vps16. At 48 h post-transfection with HA-Vps16 and Flag-UVRAG C-terminal mutants, 293T WCLs were immunoprecipitated with anti-Flag, followed by immunoblotting with anti-HA. (d) UVRAG C2 domain or C-terminal region deletion abolishes Vps16 binding. At 48 h post-transfection with HA-Vps16 together with Flag-UVRAG or its mutants, 293T WCLs were immunoprecipitated with anti-Flag followed by immunoblotting with an anti-HA antibody. (e) Schematic representation of UVRAG and its deletion mutants. ++, strong binding; +, weak binding; −, no binding. The raw data for a, c and d are shown in Supplementary Information, Fig. S6
Organismal Phenotypes in Chimeric Mice.Figure 5(A) Chimeric mice with more than 50% wild-type contribution do not exhibit wasting. Weekly weights for each of the mice were recorded from 4 wk until the mice were sacrificed. For wild-type and npc1−/− homozygous mice, the curve shown is the mean weight from three to four mice. A progressive decrease in weight of npc1 mice begins to show around 7 wk. The chimeric mice display a delay in, or even absence of, wasting depending on the amount of wild-type contribution. (B) Prevention of ataxia in chimeric mice. Mice were assessed for ataxia weekly during two 3-min trials on a balance beam (see Materials and Methods). (C) Gait of chimeric mice. Front and back paws of mice were dipped in red or green paint, and mice walked across a box lined with paper. The npc1−/− mouse displays shorter stride length and a smearing of the footprints as the paws are not as well lifted between steps. Chimeric mice with high amounts of wild-type contribution (C4.8; C4.4 not shown) exhibited a gait indistinguishable from wild-type. (D) Quantification of stride length. For wild-type and mutant controls, three mice were assessed. For chimeric mice, gait was measured prior to sacrifice at the ages noted. Error bars indicate the standard deviation. Comparison with the GFP mice demonstrates that npc1+/+, C1.4, C4.4, and C4.8 do not have significantly different stride lengths (p > 0.05) while npc1−/−, C5.2, C5.6, C2.1, and C5.8 have significantly shorter stride lengths (p < 0.001)
Formation of ATG-9 puncta in various autophagy mutants.Figure 6.Formation of ATG-9 puncta in various autophagy mutants. (A and B) In wild-type embryos, ATG-9::GFP is diffusely localized in the cytoplasm. (A) DIC image of the embryo shown in B. Insets show a magnified view. (C) ATG-9::GFP forms a large number of small, intense puncta in epg-1 mutants. (D and E) Accumulation of ATG-9::GFP puncta in unc-51(D) and epg-1 (E) mutants is independent of lgg-1. (F) The ATG-9::GFP puncta largely disappear in epg-1;epg-7 mutants. (G) ATG-9::GFP accumulates into large punctate structures in epg-6 mutants. (H) Loss of function of lgg-1 suppresses the accumulation of ATG-9::GFP puncta in epg-6 mutants. (I) Loss of function of epg-7 has no effect on the formation of ATG-9::GFP puncta in epg-6 mutants. The ATG-9::GFP puncta are smaller in epg-6;epg-7 mutants than those in epg-6 single mutants. (J-L) ATG-9::GFP accumulates into large punctate structures in epg-4 mutants (J) and the accumulation is suppressed by simultaneously depleting the activity of lgg-1 (K) or epg-7 (L). (M and N) In epg-8 mutants, ATG-9 accumulates into aggregates (M) that are suppressed by loss of lgg-1 activity (N). (O) epg-7 mutants exhibit the same distribution pattern of ATG-9::GFP as wild-type embryos. (P) Percentage of ATG-9::GFP puncta colocalized with EPG-7 and SQST-1 aggregates in indicated autophagy mutants. (Q-T) The ATG-9::GFP puncta in epg-6 (Q), epg-8 (R), unc-51 (S), and atg-18 (T) mutants colocalize with EPG-7 aggregates. (U and V) ATG-9::GFP puncta in epg-6 (U) and epg-8 (V) mutants colocalize with SQST-1 aggregates. (W and X) In unc-51 (W) and atg-18 (X) mutants, ATG-9::GFP partially colocalizes with SQST-1 aggregates.
Expression signatures of tumor endothelial and immune cells.Figure 2Top-left - tSNE plot of the Seurat clusters, boxes demarcate compared clusters. Dashed boxes and labels indicate the cell clusters that are compared in panels A-C. (A) Volcano plot of differential gene expression (DGE) between liver vascular endothelial cells in the tumor and non-tumor samples. (B) Volcano plot of DGE between mononuclear phagocytes in the malignant and non-malignant samples. (C) Volcano plot of DGE between T cells in the tumor and non-tumor samples. (D) DGE analysis between tumor mononuclear phagocytes classified by cancer type (cholangiocarcinoma in dark purple and metastasis in light purple). Wilcoxon rank-sum tests were used to generate p-values, Benjamini-Hochberg multiple hypotheses correction was used to compute q-values. Labeled dots in all panels are gene names of selected differentially expressed genes between the compared two clusters.
TGF-β1 regulates Sema3A production and NEM recruitment.Figure 6A-C Endothelial Sema3A expression is directly inhibited by VEGF and stimulated by TGF-β1 in vitro. Mouse aortic endothelial cells were stimulated with increasing doses of recombinant VEGF164 or TGF-β1 for 24 h. The expression of Sema3a and Tgfb1 was quantified by qRT-PCR and expressed as fold-change versus the non-stimulated cells. VEGF dose dependently inhibited Sema3a expression (A), but did not affect that of Tgfb1 (B), while TGF-β1 upregulated Sema3a expression in a dose-dependent fashion (C). Data represent individual values, with mean ± SEM (n = 4); **P < 0.01 and ***P < 0.001 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation; Sema3A expression upon VEGF stimulation: 40 versus 0 P = 0.0041; TGF-β1 upon VEGF stimulation: no significant differences; Sema3A expression upon TGF-β1 stimulation: 1 versus 0 P < 0.0001; 2.5 versus 0 P < 0.0001; 40 versus 0 P < 0.0001. D, E Inhibition of TGF-β1 abolishes Sema3A production and NEM recruitment in vivo. Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) VEGF levels, were treated with a TGF-β1 blocking antibody. Analyses were preformed after 1 week. (D) Immunohistochemistry for Sema3A protein on frozen muscle sections with H&amp;amp;E counterstaining. The intensity of Sema3A stain was quantified as reciprocal intensity on a scale out of 250. Scale bar = 50 μm. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: No stain, n = 10; Ctrl IgG1, n = 17; Ctrl AbαTGF-β1, n = 16; V Low IgG1, n = 23; V Low AbαTGF-β1, n = 28. **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low IgG1 versus No stain P = 0.0069; V Low IgG1 versus Ctrl IgG1 P < 0.0001; V Low AbαTGF-β1 versus V Low IgG1 P = 0.0002. D, E Inhibition of TGF-β1 abolishes Sema3A production and NEM recruitment in vivo. Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) VEGF levels, were treated with a TGF-β1 blocking antibody. Analyses were preformed after 1 week. (E) Quantification of recruited NEM (number of CD11b+ cells/mm2 of area). Scale bar = 25 μm. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: Ctrl IgG1, n = 20; Ctrl AbαTGF-β1, n = 19; V Low IgG1, n = 20; V Low AbαTGF-β1, n = 27; ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low IgG1 versus Ctrl IgG1 P = 0.0005; V Low AbαTGF-β1 versus V Low IgG1 P < 0.0001.
a, b, Representative confocal images of cilium marker acetylated tubulin from MEFs with indicated genotype subjected to 24 h serum starvation.f3a, b, Representative confocal images of cilium marker acetylated tubulin from MEFs with indicated genotype subjected to 24 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia or length of the cilia for 500 cells or 100 cells per well, respectively, in triplicate samples. ***P  0.001, two-tailed unpaired student's t-test. Similar results were observed in three independent experiments. Un, untreated; SS, serum-starved.
Figure 7(A) Flow cytometry analysis of liver myeloid subsets (CD11b+ Gr-1high, CD11b+ Gr-1intermediate, CD11b+ Gr-1-) isolated from Lyzs-Cre and p38γ/δLyzs-KO mice fed a MCD for 3 weeks or HFD for 10 weeks. Representative dot plots are shown, and bar charts show the diet-induced increase in each population as a percentage of the total intra-hepatic CD11b+ leukocyte population. Myeloid infiltrating cells isolated from livers were sorted by FACS and stain with H&amp;amp;E. Representative cells were shown next to the appropriate myeloid subsets. (B and C) Neutrophils and monocytes as a percentage of total circulating leukocytes, measured in total blood in animals fed the MCD diet for 3 weeks (B) or the HFD for 10 weeks (C). (D and E) WT mice fed the MCD diet were i.v. injected with a 1:1 mix of DiO-labeled Lyzs-Cre neutrophil and DiD-labeled p38γ/δLyzs-KO neutrophils (6x106 cells in total; n=10). One hour after injection, liver-infiltrating neutrophils were assessed by flow cytometry (D) and fluorescence micrography on liver sections (E). (F) Intravital microscopy quantification of the rolling and adhesion frequencies and rolling velocities of neutrophils recruited to venules irrigating inflamed (TNFα-injected) cremaster muscle. Data are means ± SEM. (n=5-10). *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni's post tests).
Human S100A9 inhibits HIV-1 reverse transcriptase activity in vitro in a divalent cation-dependent manner.Figure 11Schematic description of E2C and S100A9 immunopurification and elution protocol (left panel) and western blot analyses of total extracts (input) and eluated immunopurified E2C and S100A9 (right panels). Colorimetric HIV-1 RT activity assay using constant amount of HIV-1 RT (0.5ng) and performed with increasing amounts of immunopurified E2C and S100A9 eluates. Means ± SD colorimetric data obtained from duplicates of depicted independent experiments were normalized to E2C-containing conditions and shown on graphs. Each datapoint in the graph represents an independent experiment. Colorimetric HIV-1 RTase activity was performed with 10µg of immunoprecipitated and eluted E2C or S100A9 in presence of increasing MgCl2 concentrations. Means ± SD colorimetric data obtained from at least three independent experiments were normalized to E2C-containing conditions and shown on graphs.
| IgD expression modulates B cell responsiveness A | Quantification of surface IgM (top) and IgD (bottom) MFI in B cells from mice of the indicated genotypes.Figure 6A | Quantification of surface IgM (top) and IgD (bottom) MFI in B cells from mice of the indicated genotypes. Single dots represent the average MFI of 4 independent measurements per mouse (n), mean ±SD. Statistical significance was calculated by using the Kruskal-Wallis test n. s. = not significant; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. | Representative intracellular Ca2+ influx measured in Thy1.2- splenocytes, derived from mice of the indicated genotypes, upon stimulation with cHEL, monovalent sHEL (both at a concentration of 1 μg/ml) or 10 μg/ml α-mouse κLC antibody, respectively. Line diagrams below the dot plots show the overlayed MFI of the Ca2+ influx kinetics displayed in the plots above. C | Quantification of intracellular Ca2+ influx The median area under the curve of the Ca2+ influx kinetics was calculated, single dots represent data from individual mice, mean ±SD. Statistical significance was calculated by applying the Mann-Whitney-U test n. s. = not significant, ** p ≤ 0.01.
a) Western blots showing the inhibitory effect of 3-MA on autophagy in I/R.f6(a) Western blots showing the inhibitory effect of 3-MA on autophagy in I/R. Full-size blots are shown in Supplementary Fig. 13. (b) TTC-stained sections showing the infarct area after administration of 3-MA (100 nmol, i.c.v., at reperfusion) in WT and H3R−/− mice (scale bar, 5 mm), and (c) bar graph showing infarct volume (n=6-8 mice per condition; *P0.05, ***P0.001, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test). (d) TTC-stained sections (scale bar, 5 mm) and bar graph showing the infarct area after administration of thioperamide in Atg5+/−mice (n=4-6 mice per condition). (e) In cultured neurons, viability was tested by MTT assay after 3-MA (2.5 mmol l−1 at reperfusion) and thioperamide was administered in OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test). (f) In cultured neurons, viability was tested by MTT after siRNA for Atg7 and OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test). (g) Viability was tested by MTT assay on Atg5+/+and Atg5−/− MEFs after thioperamide administration after OGD/R (n=7 per condition; **P0.01, ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.
Figure 2PCA on the amino acid frequency of the last 50 residues of functional human proteins. Human protein coding sequences were downloaded from NCBI GeneBank. The terminal (-1) and the final 10 residues (-2~-10) are labeled red and blue, respectively, with the remainder marked yellow. The relative frequency of Gly (top) and diGly (bottom) in the final 50 residues of functional and aberrant human proteins. We only analyzed the last 10 residues of peptides translated from uORFs due to their inherently smaller size. The number on each graph indicates the left-tailed FDR (false discovery rate)-adjusted p-value of the extreme terminal (-1) residue. Analyses using human sequences downloaded from Uniprot database are shown in Fig. EV1C and EV1D. The relative frequency of Gly at the C-termini of indicated proteomes, i.e. mouse (Mus musculus), fly (Drosophila melanogaster), nematode (Caenorhabditis elegans), plant (Arabidopsis thaliana), yeast (Saccharomyces cerevisiae), and bacteria (Escherichia coli). Stability (mean GFP/RFP) comparisons between the wild-type protein and its mutant in which the terminal residue has been changed to Gly. Detailed information on tested proteins is presented in Table EV3. Comparisons of protein stability among wild-type protein and the mutants indicated on the right.
Figure 9In order to act as a transcriptional repressor of epithelial differentiation genes, ZEB1 binds to DNA via a dual E-box motif and recruits co-repressors like CTBP. Within the same cell, recruitment of ZEB1 by YAP and the AP-1 factors FOSL1 and JUN turns ZEB1 into a transcriptional activator of tumour-promoting genes. This activator complex which we propose here can form independently of the presence of high affinity ZEB1 motifs and likely ZEB1 is indirectly recruited to DNA by AP-1 and YAP/TEAD. It can however not be excluded that degenerated ZEB1 motifs support complex formation in a native chromatin context. While ZEB1 acts as a transcriptional repressor mainly from promoter regions, the ZEB1/YAP/AP-1 complex preferentially locates to distal regulatory regions. Together, both modes of ZEB1 action exerted in the same cell contribute to cancer aggressiveness. Orange box: ZEB1 specific E-box motif. Dashed orange box: degenerated ZEB1 motifs. Green boxes: AP‑1 and TEAD consensus motifs.
Conformation of ABCE1 in native 40S initiation complexes.Figure 3A Overall position of ABCE1 in 40S initiation complexes, here representatively shown for the human State II with eIF3j. B-D Schematic representation and structure of semi-open ABCE1 as in 80S-pre-splitting complexes (Brown et al., 2015, PDB 3JAH) (B), hybrid semi-open/closed ABCE1 as in native 40S initiation complexes (C) and fully closed ABCE1 as in in vitro reconstituted post-splitting complexes (Nürenberg-Goloub et al., 2020, PDB 6TMF) (D). Nucleotide binding sites colored in blue and bound nucleotide indicated with yellow circles (one circle per phosphate group). E Zoom into NBSI and NBSII showing bound Mg2+-ADP (in NBSI) and Mg2+-ATP (in NBSII) fit in density as obtained after focused classification on ABCE1 and refinement. F View focusing on the NBDs and the unassigned extra density (ED) reaching from the 40S via the HLH into NBSI. The ABCE1 map was low-pass filtered at 6 Å. Schematic representation highlighting the position of the ED with respect to the NBSs. G Upper panel: Position of eIF3j and ABCE1 in the crosslinked yeast 43S-PIC (43S-PIC-XL) sample. View focusing on the ABCE1-eIF3j interaction (same view as (F)): An extra density attributing to the eIF3j N-terminal region is connecting the eIF3j 6-helix bundle with NBSI of ABCE1. The map was low-pass filtered at 8 Å. Lower panel: N-terminally extended model of eIF3j (transparent blue) highlighting the position of K118, which was found crosslinked to K121 and K181 of ABCE1 (atoms colored in yellow).
Figure 1SILAC-based proteomics data identify known (red) & novel (green) hypoxia-induced proteins in SW620 cells. One sample T-test was performed. Western blotting confirmed GPRC5A as a hypoxia-induced protein in SILAC lysates. Validation of GPRC5A western blot data using siRNA. *Non-specific band of ~60kDa not depleted by GPRC5A siRNA. Confocal microscopy showing plasma membrane GPRC5A expression in hypoxic SW620 cells (scale bars: 75um). Western blotting showing GPRC5A upregulation by hypoxia in a panel of colorectal tumour cell lines. Basal & hypoxia-induced GPRC5A protein expression was decreased by HIF-1/2α depletion. Depletion of HIF-1β decreased GPRC5A protein upregulation in hypoxia. Hypoxia mimetic DMOG induced HIF-1/2α, CA9 & GPRC5A protein expression. Dual HIF-1/2α depletion reduced GPRC5A induction by DMOG. qRT-PCR demonstrating that GPRC5A mRNA was upregulated by hypoxia. GPRC5A was normalised to HPRT (error bars ± SD). qRT-PCR demonstrating that HIF-1/2α depletion decreased GPRC5A induction during hypoxia. GPRC5A was normalised to HPRT (error bars ± SD). ChIP/PCR analyses identify HIF-1α a binding to the GPRC5A promoter region containing a putative optimal HRE (error bars ± SD).
Figure 152-day-old (A) plants of the genotypes indicated grown in LDs. Scale bars: 5 cm. B-C The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A the numbers indicate independent transgenic lines. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In (B-C data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test). D-E qPCR results showing mRNA expression of CIB1, FT in the genotypes indicated. The numbers indicate independent transgenic lines. The transcript levels of CIB1 or FT in Col were set to 1.0. Samples were collected from 7-day-old LD grown seedlings at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed. F. 25-day-old plants (F) of the genotypes indicated grown in LDs. Scale bars: 5 cm. , G-H. The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in the numbers indicate independent transgenic lines. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In G-H), data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test). , I-J. qPCR results showing mRNA expression of CO, FT in the genotypes indicated. The numbers indicate independent transgenic lines. The transcript levels of CO or FT in Col were set to 1.0. Samples were collected from 7-day-old LD grown seedlings at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.
The lysosome regulates gene expression via TFEB.Figure 6(A) Chloroquine treatment inhibits mTORC1 activity in primary hepatocytes. Primary hepatocytes isolated from 2‐month‐old Tcfebflox/flox (control) and Tcfebflox/flox;Alb‐Cre(Tcfeb−/−) mice were left untreated, or treated overnight with Torin 1, U0126, or Chloroquine. Subsequently, cells were lysed and protein extracts were immunoblotted with the indicated antibodies. (B, C) TFEB mediates the transcriptional response to chloroquine and Torin 1. Quantitative PCR (qPCR) of TFEB target genes in primary hepatocytes from control (flox/flox) and Tcfeb−/− mice. Cells were treated with Chloroquine (left) or Torin 1 (right). The graphs show the relative increased expression in the treated versus the corresponding untreated samples. Values represent means±s.d. of three independent hepatocyte preparations (three mice/genotype). Student's t‐test (two tailed) *P‐value ⩽0.05.