EMBO/sd-character-level-ner huggingface dataset:
WASH inhibits Vps34 activity.Figure 4(A) WASH deficiency enhances the interaction of Vps34 with Beclin 1. WASH+/+ and WASH−/− MEFs were treated with EBSS at 37°C for 1 h and immunoprecipitated for Beclin 1. IP, immunoprecipitation. Ratio of Vps34/Beclin 1 was calculated by the Image J software and shown in the right panel. (B) WASH KO MEFs were treated with or without EBSS at 37°C for EBSS followed by kinase assays. Cells were lysed and incubated with anti‐Beclin 1 antibody to precipitate the autophagy‐related Vps34. Immunoprecipitates were separated into two equal parts, one for kinase assays and the other for input detection. RLU, relative light unit. (C) WASH deficiency enhances Vps34 activity. WASH+/+ and WASH−/− MEFs stably expressing GFP‐WIPI1 were stimulated with or without EBSS for 1 h followed by confocal microscopy (left panel). GFP‐WIPI1 dots were calculated and shown in the right panel. Scale bar, 10 μm. (D) WASH‐silenced HeLa cells were treated with or without EBSS at 37°C for 1 h, and detected as above. (E) WASH attenuates the interaction of Beclin 1 with Vps34. MEFs stably expressing vector or Flag-WASH were starved with EBSS for 1 h, followed by immunoprecipitation with anti‐Beclin 1 antibody. Ratio of Vps34/Beclin 1 was shown in the right panel. (F) WASH overexpression reduces Vps34 activity. MEFs stably expressing GFP‐WIPI1 were transfected with vector or Flag-WASH and then starved with EBSS for 1 h, followed by visualization with confocal microscopy (upper panel). GFP‐WIPI1 dots were calculated and shown in the lower panel. Scale bar, 10 μm. Data are shown as means±s.d. **P0.01 and ***P0.001. Experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page.
Figure 3Protein-protein interaction network between 29 viral proteins of SARS-CoV-2 and the host proteins is shown for 437 pairs of host-virus interactions. The red diamonds are virus proteins (NSP, non-structure protein), grey rounded rectangles are groups of proteins belonging to the same protein complex or from the same pathway. The italic text are the functional characterizations analyzed with Metascape and reference mining, circles are the high-confidence interacting proteins (HCIPs) identified from SFB-tandem affinity purification experiments. The colors of the cycle indicate the fold change of that prey calculated by SAINTexpress comparing to control SFB-TAP purifications. The size of the cycle are PSMs number identified for that prey.
a) Chemical structure of PIK-III.figf1(a) Chemical structure of PIK-III. (b) The indicated kinases were incubated with different doses of PIK-III and IC50 values were determined. (c) U2OS cells expressing GFP-FYVE were incubated with the indicated compounds for 2 h, fixed, stained with Hoechst 33342 and imaged by automated epifluorescence microscopy. GFP-positive puncta per cell were calculated and used to determine IC50 values for each compound. Data points represent values from three wells of one experiment. (d) U2OS cells expressing GFP-FOXO3A were incubated with the indicated compounds for 2 h, fixed, stained with Hoechst 33342 and imaged by automated epifluorescence microscopy. The ratio of nuclear to cellular GFP fluorescence intensity was calculated and used to determine IC50 values for each compound. Data points represent values from three wells of one experiment.
Figure 7Experimental schema. After the introduction of RV into NPCs in a proliferative condition, FGF2 was withdrawn for 2 days, and samples were collected. Confirmation of Lamin B1 overexpression by qRT-PCR (*P = 0.020, n = 3, one-sample t-test) C. Confirmation of Lamin B1 overexpression by immunocytochemistry in NPCs. Scale bar = 10 µm. D, Relative induced expression of differentiation markers upon the withdrawal of FGF2. 0 % indicates the same expression levels with control. Exogenous expression of lamin B1 inhibits the induction of genes related to neural differentiation (Tuj1, *P = 0.023; Prox1, ***P = 0.00012; NeuroD1, ** P = 0.0046; Ascl1, ***P = 0.00064; S100β, ***P = 0.000097; n = 3, one-sample t-test). E, Experimental schema. RV:EGFP-IRES-EGFP or LaminB1-IRES-EGFP was injected into the DG of wildtype mice and the brains were collected 7 days later. F, Confocal images after exogenous expression of lamin B1 in EGFP+ cells with RV;LaminB1-IRES-GFP (laminB1-OE.) Scale bar = 20 µm. G, H, Exogenous expression of laminB1 repressed neuronal differentiation in vivo. An arrowhead indicate DCX+GFP+ cell in control (G, left) and open arrowheads indicate Sox2+GFP+ cells in lamin B1-OE cells. The fraction of EGFP+ DCX+ cells was significantly reduced by lamin B1 overexpression (***P < 0.0002, n = 3), whereas the fractions of EGFP+DCX+Sox2+ cells and the EGFP+Sox2+ were increased (EGFP+DCX+Sox2+, **P = 0.0024; EGFP+ Sox2+, P = 0.058; others, *P = 0.02). Scale bar = 20 µm.
Figure 6A (Left) Detection of XBP1 mRNA splicing in WT and PDI KO HepG2 cells treated with or without 5 μM Tg for 1 h. PDI KO was verified by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean ± SEM of three independent experiments. *p < 0.05 (two-tailed Student's t-test). B (Left) Detection of XBP1 mRNA splicing in PDI KO HepG2 cells expressing PDI WT or its mutants treated with or without 5 μM Tg for 1 h. PDI expression levels were shown by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (one-way ANOVA, the post hoc Tukey's HSD test). C Detection of IRE1α, PERK and ATF6 activation by immunoblotting in PDI KO HepG2 cells expressing PDI S537A or S357E treated with 5 μM Tg for 30 min. Asterisk indicates unspecific background band. D HepG2 cells were treated without or with 5 μM Tg for 1 h. Endogenous IRE1α-immunoprecipitates were analyzed by PDI and BiP immunoblotting. E HepG2 cells expressing FLAG-tagged IRE1α were treated without or with 5 μM Tg for 1 h. An aliquot of Tg-treated cells was then washed with culture medium and chased for 4 h. FLAG-immunoprecipitates were analyzed by PDI and BiP immunoblotting. F shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 1 h. Endogenous IRE1α-immunoprecipitates were analyzed by PDI and BiP immunoblotting. G Co-immunoprecipitation of FLAG-tagged IRE1α and HA-tagged PDI in HepG2 cells expressing V5-tagged Fam20C WT or DA. H Co-immunoprecipitation of FLAG-tagged IRE1α, HA-tagged PDI WT or S357A in HepG2 cells expressing V5-tagged Fam20C. I Co-immunoprecipitation of HA-tagged PDI and FLAG-tagged IRE1αNLD in HepG2 cells. J Recombinant GST-PDI was phosphorylated by Fam20C protein for 2 h, and then used to pulldown His-IRE1αNLD protein, as visualized by Coomassie blue staining. Successful phosphorylation of GST-PDI was verified by pS357-PDI immunoblotting. K Co-immunoprecipitation of FLAG-tagged IRE1αNLD WT or its mutant C109/148/332S with HA-tagged PDI (Left) or MYC-tagged P5 (Right) in HepG2 cells. L Co-immunoprecipitation of FLAG-tagged IRE1α and HA-tagged PDI WT or its binding mutant W128I/L403W in HepG2 cells expressing V5-tagged Fam20C or not.
Proteomics analysis reveals losartan-mediated reversion of early fibrotic changes in RDEB.Figure 6Heat map of cluster analysis of protein abundances determined by label-free quantification mass spectrometry. Extracted ion currents were used to determine protein abundances, and respective intensities were log2-transformed and normalized (z-score). Samples were clustered hierarchically, and protein abundances were clustered by k-means. Cluster sizes are indicated by color code on the left. Clusters 3, 4, 5, 8, and 9 highlight losartan-induced changes. The general patterns of protein abundance in clusters 5, 8, and 9 are similar in wild-type and losartan-treated C7-hypomorphic mice and thus contain downstream targets of losartan involved in RDEB disease progression. WT, wild-type; H, hypomorphic; H + Los, losartan-treated C7-hypomorphic skin. Proteins from cluster 5 carrying GO terms related to inflammation were short-listed and analyzed on potential interactions using default settings in STRING DB (confidence score 0.4) (Szklarczyk et al, 2015). Bar graphs show abundance of selected representative proteins in cluster 5 that were normalized by losartan treatment. Shown to the left are the mean ± S.E.M. of the normalized protein abundance (LFQ intensity) of groups of individual mice corresponding to wild-type, untreated, and losartan-treated C7-hypomorphic mice as indicated in the figure. Unpaired t-test was used to calculate significance. Abundance of Lrg1: *P-value C7-hypomorphic vs. losartan-treated C7-hypomorphic mice = 0.022; abundance of serpin f2: **P-value untreated C7-hypomorphic vs. losartan-treated C7-hypomorphic mice = 0.0084; abundance of vitronectin: **P-value untreated C7-hypomorphic vs. losartan-treated C7-hypomorphic mice = 0.0094. Right, validation of proteomics analysis by Western blotting of independent biological replicates. Representative Western blots of skin lysates from wild-type, untreated C7-hypomorphic, and 7-week losartan-treated C7-hypomorphic mice not used for proteomics. Blots were probed with antibodies against proteins as indicated. Erk1/2 and β-tubulin served as loading controls. The analysis shows that there is a good correlation between proteomics data and abundance detected by Western blotting (n = 3 per group).
Figure 6Northern blot experiments (A-C) assessing GlmY* half-life under GlcN6P replete (+GlcN) and depletion conditions (-GlcN). Transcription was stopped by addition of rifampicin and samples were harvested at indicated times for Northern analysis. A Analysis of the ∆glmS strain Z1126. The GlcN6P depleted culture was split 8 min after rifampicin addition and one of the sub-cultures was resupplied with GlcN (indicated by arrow). B The ∆glmS ∆rapZ double mutant Z1127 was tested. C Semi-logarithmic plots of GlmY* decay for half-life determination. D EMSA experiments addressing the role of GlcN6P for GlmY*/RapZ interaction. Radiolabelled GlmY* was incubated with incremental concentrations of RapZ (left panel) or RapZ-CTD (right panel) in absence or presence of 7.5 mM GlcN6P. Binding reactions were separated on native PAA gels and analyzed by phospho-imaging. The RapZ/GlmY* complex is indicated by an arrow. E EMSA following incubation of GlmY* with 1200 nM RapZ in presence of various GlcN6P concentrations ranging from 0 (lane 2) to 8 mM (lane 10). The fraction of GlmY* remaining in the gel pocket is marked with an asterisk. The RapZ/GlmY* complex is indicated by an arrow. F EMSA following incubation of GlmY* with 1200 nM RapZ in absence or presence of 7.5 mM of the indicated metabolite. The RapZ/GlmY* complex is indicated by an arrow.
Figure 3A Schematic representation of the experimental strategy for the RIP-seq pipeline. Representative Western blot showing the FXR2P pull-down. FXR2P was precipitated from WT and Fxr2 KO mice brain extracts using the 1G2 antibody (Fernandez et al, 2015) and analyzed by Western blotting. Input, 10%. PSD-95 mRNA was used as control to verify the specificity of the RIP-seq. B PSD-95 mRNA had lower read counts in Fxr2 KO samples (n=3) compared to WT samples (n=3) (+ adjusted p < 0.1, two-tailed Student's t-test). Data are presented as mean ± SEM. C Enrichment analysis of the 14'809 transcripts shows 488 genes (red) that pass the FDR threshold (adjusted p-value cut-off at 0.1). D Left, gene ontology of the 488 FXR2P candidate mRNAs across the top 10 cellular components. Right, biological processes specific for glutamate are highlighted. -Log10 q-values were normalized according to the maximum q-value in the list of gene ontologies. The red dashed line indicates the threshold for significant enrichment in these gene ontologies. E Left, FXR2P binding motif sequence as described by Ray et al (2013). Right, distribution of match scores between FXR2P target and non-target mRNAs. FXR2P target mRNAs show on average higher match scores than FXR2P non-target mRNAs (Kolmogorov-Smirnov test, p < 0.001).
| Correlation analysis of protein and transcript expression levels.Figure 3Global correlation analysis of proteomes vs proteomes and transcriptomes vs transcriptomes across human tissues. It is apparent that proteomes correlate stronger across tissues than transcriptomes. Co-inertia analysis of transcriptome and proteome levels of all 29 tissues (arrow base: transcriptome; arrow head: proteome) showing that the information carried by transcriptomes and proteomes were closer to each other in the same than across different tissues. Grey lines are used to aid identifying tissue names for the respective arrows. Shaded areas highlight tissues that are related by their molecular profiles. Average cellular compositions of tissues highlighted in panel (B) showing that the molecular similarities in their transcriptomes and proteomes are driven by similarities in cell types.
Q depleted and quantitatively modified cell culture conditions.Figure 2A Cell culture timing schedule. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine). B Corresponding queuosinylation and methylation levels, as determined by Northern blot using a tRNAHisGUG probe and bisulfite sequencing of tRNAAspGUC. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine). C Representative metaplot of SF cells. 25-32 mer ribosome footprint reads were summed across all annotated reading frames with the AUG staring codon at position zero. In-frame 27-30 mer ribosome footprints showing the correct periodicity. D Representative metagene plot of 27-30 mer ribosome footprints in S and SF culture conditions for two biological replicates (rep1 and rep2). Data information: SF (serum free medium), q (queuine)
Figure 1Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. Immunodetection of the indicated proteins after BNGE Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. Immunodetection of the indicated proteins after BNGE Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. (C) in-gel activity for CI and (D) CIV (shown is a representative gel from two technical and two biological replicates). BNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. Immunodetection of the indicated proteins BNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. (G) in-gel activity of CI and (H) CIV (representative gel from two technical and three biological replicates). I, 2D BNGE/SDS electrophoresis: 1st dimension with digitonin (Dig) and 2nd dimension with SDS, followed by immunoblotting with the indicated antibodies to identify the proteins detected by the commercial anti-SCAF1 antibody. Asterisks indicate missing bands in scaf1Δ1/Δ1.
NMN addition and Nmnat overexpression rescue lysosomal function.Figure 4(A) Lysosomal acidification is impaired in p32KO MEFs. Representative images of WT and p32KO MEFs, stained with dextran-Oregon Green (488) and dextran-TMRM. Scale bar, 5 µm. Addition of 1 mM NMN for 48 h to p32KO MEFs rescued lysosomal acidification, and 1 mM CAP treatment of WT MEFs for 48 h decreased lysosomal acidification. Dextran-Oregon Green is quenched under acidic pH, and thus an increased green:red ratio denotes impaired lysosomal acidification. The average ± SEM of the green:red ratio in at least 30 cells in two independent experiments is presented in the plot (normalized to WT green/red = 1). Statistical significance was assessed by Student's t-test, *p < 0.05, ***p < 0.001. (B) Two Nmnat3 constructs: Nmnat3 (full) with a mitochondrial-targeting sequence (MTS), and Nmnat3(v1) without MTS. (C) Both these constructs were effectively expressed in p32KO MEFs. After transfection, lysates were subjected to western blot analysis with anti-Flag and anti-Nmnat3 antibodies. GAPDH was used as an internal control. One representative experiment out of three shown. (D) Nmnat3 (full) was localized in mitochondria, but Nmnat3(v1) did not co-stain with MitoTracker red in p32KO MEFs. Scale bars, 10 µm. The magnified image is shown on the right. (E) Nmnat3(v1) overexpression in p32KO MEFs rescued lysosomal function. LysoTracker Red-stained p32KO MEFs were quantified by flow cytometry. Error bars are presented as mean ± SEM of 4 independent experiments. Statistical significance was assessed by Student's t-test, ***p < 0.001.
BI‐1 controls autophagy activation in vivo in Drosophila melanogaster.Figure 6(A) The expression of dBI‐1 was knocked down in Drosophila melanogaster. Then, relative expression levels of dBI‐1 mRNA were monitored by semiquantitative PCR. Actin levels were monitored as control. (B) (B) LC3 levels were monitored in control (Da‐Gal4>huLC3:GFP) or dBI‐1 RNAi larvae (Da‐Gal4>huLC3:GFP, Dcr2, dBI‐1i) under basal or fasting conditions. Then, huLC3-GFP levels were analysed by western blot. In addition, dBI‐1i larvae were treated with 100 μM SP600125 (added in the growing media). (C) Left panel: the presence of LC3‐positive vesicles (white arrowheads) was monitored by confocal microscopy in control (Da‐Gal4>huLC3>GFP) and dBI‐1 knockdown pupae (Da‐Gal4>huLC3:GFP, Dcr2, dBI‐1i) after 6 h of puparium formation. The organization of actin cytoskeleton was monitored by staining with phalloidin (Ph, red). Nucleus was stained with Topro (blue). Scale bar: left 20 μm and right 11 μm. Right panel: overexpression of dBI‐1 (dBI‐1EY03662) delays salivary gland degradation. Overexpression of dBI‐1 was confirmed by semiquantitative RT-PCR. Actin levels were monitored as loading control. Right panel: wild‐type control and dBI‐1EY03662 pupae were analysed at 14 h after puparium formation. Superficial and internal confocal planes of the cells are presented. Scale bar: 40 μm. (D) Wild‐type control and dBI‐1EY03662 larvae were cultured in fasting conditions for different periods of time and fat body stained with lysotracker (red) and Hoechst (blue). Then, lysosomal content in the fat body was visualized by epifluorescence microscopy. The percentage of cells presenting lysotracker‐positive stain is indicated. Scale bar: 50 μm. (E) Control or dBI‐1 knockdown adult flies were exposed to nutrient starvation and then animal viability was monitored over time for several days. In all, 100 individuals were monitored in each condition. Data represent mean and standard error (N=3). Two‐way ANOVA was used to analyse statistical significance between groups. (F) Second instar dBI‐1 RNAi or control larvae were grown in food supplemented with 25 μg/ml Tm dissolved in DMSO or 0.5% DMSO as control. The number of individual reaching the adult fly stage was evaluated. Mean and standard error are presented (N=3), **P0.01.
Lactobacillus acidophilus NCK2187 and its SlpA protect intestinal barrier function and prevent dysbiosis in pathogenic T‐cell‐induced colitisColitis was induced in B6 Rag1−/− mice as described in Fig .Figure 4Colitis was induced in B6 Rag1−/−mice as described in Fig .A, B Colonic expression of tight junction‐associated genes Cldn3 and Ocln, determined by RT-PCR relative to 18S rRNA (A), were used as measures of epithelial barrier integrity. Sham adoptive transferred B6 Rag1−/−mice (white bars) were used as baseline controls in some cases. n = 5 mice/group. Data represent three individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice. Colitis was induced in B6 Rag1−/−mice as described in Fig .A, B Passive transepithelial absorption of FITC‐dextran (B), was used as measures of epithelial barrier integrity. Sham adoptive transferred B6 Rag1−/−mice (white bars) were used as baseline controls in some cases. n = 5 mice/group. Data represent three individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice. Colitis was induced in B6 Rag1−/−mice as described in Fig .C UniFrac analyses were used to calculate distances between the microbial communities of the different samples (week 7), and three‐dimensional scatter plots were generated by using principal coordinate analyses (PCoA). Gray dots = CD4+CD45RBhiT cells + PBS; yellow dots = CD4+CD45RBhiT cells + NCK56; green dots = CD4+CD45RBhiT cells + NCK2187; blue dots = CD4+CD45RBhiT cells + SlpA; red dots = CD4+CD45RBhiT cells + Tregs. Each dot represents the fecal microbiota data of an individual mouse. Colitis was induced in B6 Rag1−/−mice as described in Fig .D Changes in the relative abundance of different phyla were also analyzed. See Supplementary Table S4 for results of statistical analyses among the different groups. Dark blue = Actinobacteria; green = Bacteroidetes; red = Proteobacteria; yellow = Verrucomicrobia; aqua = Firmicutes; purple = Tenericutes; black = others. Colitis was induced in B6 Rag1−/−mice as described in Fig .E Comparison of microbial communities at family or order levels. The heat map depicts the relative value in individual mice. n = 5-6 mice/group.
Sept2 does not affect mitochondrial Myo2 localization and does not require actin dynamics for interacting with Drp1.Figure 6(A) Immunofluorescence microscopy analysis of P-MLC (green) recruitment to mitochondria (red, Tom20) in mock treated and Sept2 depleted HeLa cells. Insets show twofold enlargements. Scale bar: 10 µm. (B) Colocalization of P-MLC with mitochondria was assessed with Icy software and showed no difference between mock and Sept2 depleted cells. (C) Mock treated or Sept2 depleted Drp1 -/- MEF cells were labeled with mitotracker orange (red), treated for the indicated amount of time with 2µM FCCP and stained for actin with phalloidin (green). Insets are enlarged twofold. (D) Immunofluorescence microscopy analysis of the actin binding proteins Arp3 and cofilin (green), colocalized with cytocrome c (red) and cortactin (green), colocalized with Hsp60 (red) in mock treated or Sept2 depleted Drp1 -/- MEF cells. Scale bars: 10 µm. (E) Cytosol and crude mitochondria fractions were prepared from mock treated or Sept2 depleted Drp1 -/- MEF cells and analyzed by western blotting with the indicated antibodies. (F) HeLa cells were mock treated or treated with cytochalasin D and subjected to Sept2 immunoprecipitation. Immunoprecipitates were analyzed by western blot for Drp1 and Sept2, showing that Drp1 co-immunoprecipitation with Sept2 is not affected by cytochalasin D treatment.