EMBO/sd-character-level-ner huggingface dataset:
Ame1 and Mif2 share overlapping Mtw1c binding motifs.Figure 2GST-Mif2_1-41 was immobilized on GSH Sepharose beads and incubated with Mtw1-Nnf1 complex (MN) in the presence of increasing concentrations of Ame1_1-20 peptide (lanes 4-9). GST-Ame1_1-30 served as control (lane 2 and 3). After washing, bound complexes were eluted by adding SDS sample buffer. Protein bands in Coomassie stained gel are highlighted in the indicated color. Integrated density of Mtw1 bands was quantified using ImageJ and correlated to GST-Mif2 amounts in the same lane. Binding of FITC-Mif21-41 to MN was directly determined in a fluorescence polarization (FP) assay. This interaction at EC50 was than competed with increasing concentration of Ame11-20 unlabeled peptide. Both experiments were performed in triplicates of which the mean and standard deviation is shown. Multiple sequence alignment of the N-termini of Mif2 and Ame1 from distantly related yeast species. Conserved residues are highlighted. A dashed box indicates a conserved consensus sequence of RxxG. Equivalent point mutations replacing Arg11 in Mif2 or Arg10 in Ame1 with Alanine prevent binding to the MN complex. Mif2 and Ame1 wild-type N-termini serve as a control, protein bands are highlighted in the indicated color.
Regulation of endogenous TFEB by mTOR and Rags.Figure 3.(A) The indicated cell lines were incubated in medium containing DMSO or 250 nM Torin-1 for 1 h. Cells were then lysed and subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB) and actin. (B) HeLa cells were treated as indicated in A, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from different fractions were subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB), Lamp1, or Histone H3. (C) Immunofluorescence confocal microscopy showing nuclear and lysosomal localization of endogenous TFEB upon treatment of HeLa cells with Torin-1 as indicated in A. (D) HeLa cells were starved in serum- and amino acid-free medium (starvation) for 3 h and analyzed by immunoblotting with antibodies against TFEB. (E) HeLa cells were incubated in normal medium (control) and serum- and amino acid-free medium (Starvation) for 4 h or starved for 4 h followed by restimulation with amino acids (starvation + amino acids) for 30 min and analyzed by immunofluorescence with antibodies against TFEB (endogenous TFEB is shown in green) and Lamp1 (red). The region within the dotted box is magnified in the insets. (F) HeLa cells were nucleofected with the indicated Rag-expressing plasmids. 18 h later, cells were lysed, and RagB/D heterodimers were pulled down using glutathione-Sepharose beads. Proteins bound to the beads were analyzed by immunoblotting with antibodies against TFEB and GST (used to detect endogenous TFEB and Rag proteins, respectively). Bars, 10 µm.
Supplementation of dTMP alleviates dTDP/dTTP levels and rescues filamentation.Figure 5Distribution of cell length of WT and mutant strains when grown in M9 medium supplemented with amino acids (gray) or with both amino acids and 1mM dTMP (pink). WT, W133V and V75H+I155A strains were grown at 42°C while I91L+W133V and V75H+I91L+I155A mutants were grown at 40°C. 1mM dTMP largely rescues filamentation of mutant strains (* indicates the median cell lengths were significantly different, Mann-Whitney test, p-value <0.001). The central band in the box plots represents the median of the distribution, the box ends represent the 25th and 75th percentile, the whiskers represent the 10th and 90th percentile, while the dots represent the 5th and 95th percentile. Data was usually obtained from 2-3 biological replicates. The number of cells used to derive the boxplot distributions in the different panels range usually between 150 to 300 (please refer to Figure 5 source data for exact number of cells for each dataset). Abundances of selected nucleotides in I91L+W133V mutant when grown for 4 hours at 40°C in M9 medium supplemented with both amino acids and 1mM dTMP. Metabolite levels were normalized to those of WT grown under similar conditions. dTDP and dTTP levels recover and are now comparable to dTMP levels. Error bars represent SEM of 3 biological replicates. The dashed line in represents value of 1 for WT. Mutant V75H+ I91L+I155A grows very poorly (in terms of colony forming units, cfu) on a minimal media agar plate supplemented with amino acids (M9+AA) at 40°C. Supplementation of additional dTMP increases the cfu by several orders at the same temperature, while supplementation with both pyrimidine (dTMP) and purine (GMP) allows it to grow as good as WT. In comparison, WT was grown at 42°C under different supplementation conditions. In all cases, cultures were 7-fold serially diluted for the next spot. The three rows (two rows for WT) in each condition represent biological replicates. Comparison of growth rates of WT and mutant DHFR strains at 42°C (40°C for I91L+W133V and V75H+I91L+I155A) in minimal medium that is supplemented with amino acids and/or 1mM dTMP. Except for W133V and to a lesser extent for V75H+I155A, the effect of dTMP on growth rates is only modest. Error bars represent SEM of 3 biological replicates.
Figure 4(A MCF10A WT or two independent clones for MAVS KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. MCF10A WT or two independent clones for MAVS, RIG-I KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, (B cells were harvested for real-time quantitative PCR instead of immunoblotting. IFNB1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates. C, MCF10A WT or two independent clones for RIG-I KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. D, MCF10A WT or two independent clones for MDA5, KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. MCF10A WT or two independent clones for MDA5 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later E) cells were harvested for real-time quantitative PCR instead of immunoblotting. IFNB1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates. F) MCF10A WT or two independent clones for IRF7 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.
depletion on co-translational quality control in esophageal cancer cellsA.Figure 7A. Immunoblot of individual clones of KYSE150 cells in which HSPH1 was disrupted by CRISPR/Cas9 targeting (KYSE150HSPH1-/- cells). B. Effect of disrupting HSPH1 on stress sensitivity. KYSE150 and KYSE150HSPH1-/- cells were exposed to heat stress (45 oC) (means +/- SD, n = 3). C. KYSE150 and KYSE150HSPH1-/- cells were exposed to the indicated stress conditions and cell viability was assessed by staining dead cells with trypan blue. Graphs represent means +/- SD, n = 3, numbers above bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli. D. KYSE150 and KYSE150HSPH1-/- cells, either naïve or subjected to the aTT temperature shifting scheme, were exposed to the indicated temperature and cell lysates were separated by sucrose density gradient centrifugation. K48-linked polyubiquitin was detected by immunoblotting of gradient fractions (left panel). Signals were quantified and displayed as a line graph (right panel, means +/- SEM, n = 2). The red boxes highlight the pronounced differences in polysomal polyubiquitylation between KYSE150 and KYSE150HSPH1-/- cells. E. Whole lysates of cells KYSE150 and KYSE150HSPH1-/- cells exposed to the indicated temperature conditions (aTT scheme and heat shock) were fractionated into soluble and insoluble fractions as described in Materials and Methods. Insoluble fractions were assayed for the level of K48-linked polyubiquitin (left panel). Total levels of HSPA1 and HSPH1 are shown for reference. K48 ubiquitin signals were quantified and displayed in a bar graph (means +/- SD, n = 4). F. Lysates of cells KYSE150 and KYSE150HSPH1-/- cells exposed to the indicated temperature conditions (aTT scheme and heat shock) were obtained and polysomes were isolated by centrifugation through a 30% sucrose cushion. Whole cell lysates (left panel) and the polysomal pellets (right panel) were assayed by immunoblotting for the levels of HSPH1, HSPA1 and proteasome subunits.
Loss of Zuc and Gasz impacts the cellular localization of Armi and Piwi in OSCs.Figure 1Northern blotting shows that Gasz depletion (Gasz KD) led to the accumulation of flam-piRNA precursors but reduced the level of mature idefix-piRNA arising from the precursors, as in the case of Zuc depletion (Zuc KD). U6 snRNA was detected as a loading control. Western blotting showing the protein levels of endogenous Armi, Piwi, SoYb, Yb, Vret, and Gasz in total OSC lysate (total), the mitochondrial fraction (mito), and the cytoplasmic fraction after mitochondrial isolation (cyto). β-Tubulin (β-Tub) and HSP60 were detected as markers for the "cyto" and "mito" fractions, respectively. Left: Western blotting comparing the abundances of Armi and Piwi in total OSC lysate (total) and the mitochondrial fraction (mito) before (control) and after (Zuc KD) Zuc knockdown in OSCs. Relative mito-localization intensity (*) shows the amount of proteins in "mito" normalized by the amount of proteins in "total" and HSP60. Right: Immunofluorescence shows that the Armi signal (green) is strongly overlapped with the mitochondrial signal (red) in Zuc-depleted OSCs (Zuc KD). Armi is localized to Yb bodies in control OSCs (control). The scale bar represents 5 μm. DAPI (blue) shows the nuclei. Left: Western blotting comparing the abundances of Armi and Piwi in total OSC lysate (total) and the mitochondrial fraction (mito) before (control) and after (Gasz KD) Gasz depletion in Zuc-depleted OSCs (Zuc KD). Relative mito-localization intensity (*) shows the amount of proteins in "mito" normalized by the amount of proteins in "total" and HSP60. Right: Immunofluorescence shows that the Armi mitochondrial signal (green) found in Zuc-depleted cells (Zuc KD/control) mostly disappeared after additional Gasz depletion in the cells (Zuc KD/Gasz KD), but was found at Yb bodies. Mitochondrial signal is shown in red. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.
Figure 6Representative confocal immunofluorescence images of HA-tagged MIC19 variants with MitoTracker staining in U2OS cells. All MIC19 variants examined carried the S6W,T7P mutation. The fluorescence intensities along the dashed lines are shown as line profile graphs on the right . Scale bar = 20 µm. Fractionation analysis of wild-type HA-MIC19 in U2OS cells treated with IMP-1088 and with or without inhibition of ZYG11B or KLHDC2. The normalized relative abundance of cytosolic HA-MIC19/tubulin is indicated under each lane in red. MitoTracker staining of IMP-1088-treated U2OS cells with or without inhibition of ZYG11B or KLHDC2. The complete set of images containing MIC19 localizations is shown in Fig. EV4C. Scale bar = 20 µm. Proposed model. CRL2-operated "double-end" inspections safeguard the mitochondrial localization of MIC19.
Figure 2A, qRT-PCR (left) and Western blot (right) analyses of H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) for 24 h. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01. B, Chromatin immunoprecipitation (ChIP) assay was carried out with anti-H2Bub1 antibodies in H1299 cells (left) or 293T cells either untreated or treated with 20 μM erastin for 24 h (right). The intergenic region was used as a negative control for the occupancy of H2Bub1. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01. C, Intracellular GSH levels were examined in H1299 cells treated as indicated, and bar graphs are shown. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01. D, H1299 cells transfected as indicated were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20). E, Surviving cells from the assay shown in (D) were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01. F, GO analysis with the genes downregulated in H2BK120R (black) or RNF20-sepecific siRNA (siRNF20) (red) transfected 293T cells by employing a previously reported microarray data Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01. G, Affected metal ion-binding genes were selected and subjected to cluster analysis. H, Labile iron levels were assessed by flow cytometry with a standard method in H1299 cells. I, Labile iron levels examined were quantified. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.
Requirements for in vitro uptake.Figure 4Requirements for in vitro uptake. Standard uptake reactions were performed (60 min) (A) at different temperatures, or in the presence of different concentrations of (B) cytosol, (C) ATP, or (D) MgCl2. Luciferase uptake was determined as described in the legend to Fig. 3 A. In B, titration curves for four independent cytosol preparations are shown. The maximal uptake signal of each titration curve was set at 100%. In D, the sample drawn as 0 mM MgCl2 contained 10 mM EDTA to chelate free magnesium in the reaction.
Figure 5A-C. The IFT-interaction domain is required for Ccdc108 multiciliogenesis function in Xenopus embryos: (A) impaired multiciliogenesis, (B) defective apical migration/docking of centrioles, and (C) reduced enrichment of apical F-actin. Embryos at stage 27 were fixed and stained with the acetylated tubulin (A; Ac-tub; green), Cep164 (B; purple) and Cetn1 (B; green) antibodies or phalloidin (purple). mRNA of a membrane-bound form of GFP was co-injected with each morpholino to indicate targeted cells (A,B). Typical D-SIM images (A,B) and confocal images (C) are present. Embryos were treated as described in Figure 1C and expressed mGFP or GFP-Ccdc108 mutants. Plots show results from three independent experiments. (A) > 70 MCCs from 6 embryos, and (C) > 70 MCCs from 6 embryos for each condition. Unpaired two-tailed t-test was performed (*** p<0.001; **p<0.01). Mean ± s.d. values are presented.
Autophagy induction reduces Wnt/β‐catenin signalling.Figure 6(A) Western blotting showing that autophagy was induced after 2 h starvation, as evidenced by decreased p62 protein and increased LC3‐II protein expression. β‐Catenin protein expression decreased over a 24‐h period of starvation in HT29 cells. (B) Autophagy decreased following Atg7 knockdown as shown by increased p62 and decreased LC3‐II protein expression. β‐Catenin protein expression increased following Atg7 siRNA. Reduction of LC3 puncta with Atg7 siRNA was confirmed by immunofluorescence and is shown in Supplementary Figure S2B. (C, D) Reduction of TopFlash activity in HT29 cells after 24 h of autophagy induction using (C) starvation or (D) 100 nM mTOR inhibitor PP242 (C and D, mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). Autophagy induction was confirmed by western blotting and is shown in Supplementary Figures S2C and S2D). (E, F) qRT-PCR shows reduction of Wnt‐target gene expression (E) Axin2 (*P=0.017) and (F) Cyclin D1 (**P=0.0036) after 8 h starvation (mean±s.e.m., three (Axin2) or six (Cyclin D1) independent experiments performed in triplicate). (G-I) Inhibition of Wnt‐induced TopFlash activity by autophagy induction in (G) HCT116 β‐cateninWT/− cells (mean±s.e.m., three independent experiments performed in triplicate, **P0.01) and (H) RKO cells (mean±s.e.m., three independent experiments performed in triplicate, *P0.05). (I) Reduction of Wnt3a‐induced Cyclin D1 gene expression RKO cells after 12 h treatment with autophagy induction using starvation or PP242 (mean±s.e.m., two independent experiments). (J, K) TopFlash activity following Atg7 knockdown in (J) HT29 cells (mean±s.e.m., four independent experiments performed in triplicate, *P=0.0488) and (K) RKO cells with 24 hWnt3a treatment (mean±s.e.m., three independent experiments performed in triplicate, *P=0.0282). (L) A representative western blot of RKO cells following Wnt3a treatment and Atg7 siRNA. (M) Relative β‐catenin mRNA levels did not change after 8 h starvation by qRT-PCR (mean±s.e.m., three independent experiments performed in triplicate). (N) Western blot showing prevented β‐catenin protein degradation during starvation in the presence of lysosomal autophagy inhibitors chloroquine (10 μM) and bafilomycin A1 (100 nM) compared to starvation alone. (O) Inhibition of autophagy using wortmannin (50 nM) prevented β‐catenin protein degradation during starvation. (P) Western blot analysis of RKO cells expressing a myc-tagged β‐catenin mutant (S33A, S37A, T41A, S45A) resistant to proteasomal degradation (myc-β‐cateninAAAA). RKO cells were subject to nutrient starvation for 2, 8 and 24 h, and autophagy induction was confirmed by increased LC3‐II and decreased p62 protein expression. Both endogenous β‐catenin and myc-β‐cateninAAAA protein expression decreased during starvation. (Q) Proteasome inhibition with MG132 (10 μM) did not prevent starvation‐induced decrease in β‐catenin protein expression.Source data for this figure is available on the online supplementary information page.
Anti-apoptotic Activities of the vBcl-2 Mutant Proteins.Figure 5NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A), NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A), or for apoptosis by TUNEL staining (B) NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A), or for apoptosis by TUNEL staining (B) or for the caspase 3 activation using flow cytometry (C). Apoptotic cells in (B) were counted under high power magnification (60×magnification). Mock, untreated condition. Data represents mean±SEM of combined results from three independent experiments. Scale bars, 100 µm (A), 5 µm (B). **, P<0.0001 versus vector cells.
Figure 2A, Representative histograms and quantification of mean fluorescence intensity (MFI; A) in OT-II WT and CCR5‑/‑ lymphoblasts expanded in IL-2 or IL-15, as specified. Data shown as mean ± SEM (n ≥3). B. the percentage of cells positive for the indicated memory markers (B) in OT-II WT and CCR5‑/‑ lymphoblasts expanded in IL-2 or IL-15, as specified. Data shown as mean ± SEM (n ≥3). C-F. IL-2- (C, D) and IL-15-expanded lymphoblasts (E, F) were restimulated with indicated concentrations of OVA323-339; cell proliferation (thymidine incorporation into DNA; C, E) and IL-2 production (by ELISA; D, F) were measured after 72 h. Data are presented as mean ± SEM (n = 5).
Asymptomatic COVID-19 patients present less inflammatory monocyte, activated neutrophils and muted inflammatory response A.Figure 5A. Flow cytometry acquisition of patient whole blood was performed and number of mature and immature neutrophils were compared between asymptomatic (n=16) and symptomatic (n=52) COVID-19 patients and healthy controls (n=17). Data are presented as Mean ± SD *P<0.05; **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison). B. Mass cytometry was performed on PBMCs obtained from acute asymptomatic (n=19) and acute symptomatic (n=37) COVID-19 patients and healthy donors (n=10). Monocytes were characterized based on their CD14 and CD16 expression. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison). C. Classical, intermediate and non-classical monocytes were further defined in acute asymptomatic (n=19) and acute symptomatic (n=37) COVID-19 patients and healthy donors (n=10) with inflammatory marker CD169. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison). D. Expression data of genes associated with inflammatory monocytes, activated neutrophils and pro-inflammatory cytokines were compared between acute asymptomatic (n=30) and asymptomatic (n=26) COVID-19 patients and were represented by ratio with respect to expression in asymptomatic patients. Bar graphs show DEGs with FDR adjusted P value < 0.05 and fold change values are represented in the log2 scale. E. Pro-inflammatory associated immune mediator levels in plasma fraction samples from first collection timepoint during hospital admission were quantified with 45-plex microbead-based immunoassay. Immune mediator levels were compared between asymptomatic patients (n=48) and symptomatic patients stratified by COVID-19 severity (mild, n=61; moderate, n=43; severe, n=68). Immune mediator levels for healthy controls (HC) (n=23) are indicated by the black dotted line. Patient samples with concentrations out of measurement range are presented as the value of Limit of Quantification (LOQ). Data are presented as Mean ± SD *P<0.05; **P<0.01; ***P<0.001 (One-way ANOVA with post hoc t test).
Figure 3A Schematic representation of the Dm reporters used in panels (B,C). B,C Tethering assay using the F-Luc-5BoxB reporters shown in panel (A) and λN-HA-tagged GW182, AGO1 and NOT1 in S2 cells. F-Luc activity and mRNA levels were normalized to those of the R-Luc transfection control and analyzed as described in Fig 2A and B. Quantification of the corresponding northern blots is shown in Fig EV2J. D,E Tethering assay with the indicated reporters containing a F-Luc(Leu)-V5 fusion in which all in-frame methionines were replaced with leucine. The corresponding western and northern blots are shown in Fig EV2K-N.F Schematic representation of the reporters used in panels (G,H). F Schematic representation of the reporters used in panels (G,H). G,H Tethering assay using the R-Luc-6xMS2 reporters shown in panel (F) and HA-MS2 tagged TNRC6A-SD, AGO2 and NOT1 in HEK293T cells. R-Luc activity and mRNA levels were normalized to those of the F-Luc transfection control and analyzed as described in Fig 2C and D. The corresponding quantification of mRNA levels is shown in Fig EV3F. In all panels, bars represent mean values and error bars standard deviations from at least three independent experiments.