EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Isolated mitochondria were incubated with radiolabelled Mcp3 precursor protein for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. The arrowhead marks an additional band of the size of the cleaved N-terminus of Mcp3. p and m, precursor and mature forms, respectively. (B) Import of Mcp3 is dependent on the receptor Tom70. Mitochondria isolated from wild-type or tom70/71Δ cells were incubated with radiolabelled Mcp3 or pSu9-DHFR (as control) for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. p and m, precursor and mature forms, respectively. I, 20% of radiolabelled precursor protein used in each import reaction. Bands corresponding to the mature (m) form were quantified. Import into wild-type mitochondria after 15 min was set to 100%. The mean with standard deviations is depicted (n=3; SD). (C) Reduced steady state levels of HA-Mcp3 in cells lacking Tom70. Crude mitochondria isolated from the indicated cells were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, AAC as control substrate of Tom70, and Fis1 as a loading control. HA-Mcp3 levels were quantified in relation to Fis1 levels and the levels in WT cells were set to 100%. The bar diagram shows the mean with standard deviation (n=3; SD; *, p < 0.05). (D) Import of Mcp3 is dependent on Tom40. Mitochondria isolated from wild-type and tom4025 cells were incubated with radiolabelled Mcp3 for the indicated time periods. Next, mitochondria were reisolated and analysed by SDS-PAGE and autoradiography as described in (B). As a control Ugo1 was imported into the same mitochondria. Organelles were reisolated and analysed by BN-PAGE and autoradiography. The band corresponding to the Ugo1 dimer at around 150 kDa was quantified. The mean with standard deviation is shown (n=3; SD). (E) Steady state levels of HA-Mcp3 are lower in cells harbouring a mutant TOM40 allele. Crude mitochondria from wild-type and tom40-25 cells were obtained and analysed as described in (C). Immunodecoration was performed with antibodies against the HA-tag, Tom40, and Fis1 as loading control.
Knockdown of MPPβ leads to full‐length PINK1 accumulation at the outer mitochondrial membrane.Figure 4(A) Mitochondria from CCCP‐treated or siRNA‐transfected HEK293T cells were incubated with increasing concentrations of proteinase K (PK). (B) Mitochondria from CCCP‐treated or MPPβ knockdown cells were incubated ±0.1 M Na2CO3 before supernatant (S) and pellet (P) were separated by centrifugation. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA; WB, western blot.
Figure 4(A-D) Knockdown of Wip1 expands the expression of dorsal mesodermal markers. Embryos injected dorsally with Co MO (40 ng) or Wip1 MO (40 ng) were subjected to in situ hybridization at stage 10.25. Embryos are shown in dorso-vegetal views with dorsal to the top. Scale bar, 150 μm. (E-I) Morphological phenotypes of Wip1 morphants. Embryos were injected radially in the marginal zone with Co MO (80 ng), Wip1 MO (80 ng), wt Wip1 (1 ng) and Wip1(D277A) mRNA as indicated and cultured to tadpole stages. Embryos are shown in lateral views with anterior to the left. Un Co, uninjected control. Scale bar, 1 mm. (J) Quantification of the phenotypes shown in (E-I). Severe defects include microcephaly, shortened and kinked body axis, and no eye. Mild defects indicate normal body axis with malformed eyes.
Figure 2A) The RG loopCENP-A binding site of CENPC-CT is shown. The backbone structure of the CENPC-CT is presented in a cartoon model, which is colored in magenta, except for the pink canonical CENPC motif (aa 655-675). The cryo-EM density map of CENPC-CT is overlaid on the cartoon model. The CM downstream region beyond Y675CENP-C is indicated by a dotted box. Key residues involved in binding to the C-terminal tail of CENP-A are indicated as stick models. The cartoon models of CENP-A and histone H4 are shown in pale green and pale blue, respectively. B) Alignment of sequences around the CENPC motif region of CENP-C from various species: Gg, chicken; Hs, human; Mm, mouse; and Xl, frog. The CENPC motif and the CM downstream region are depicted by pink and purple boxes, respectively, in the sequence alignment. Schematic diagram of chicken CENPC-CT wild-type (CENPC-CT WT), CENPC-CT, in which PSG residues in the CM downstream region were substituted with AAA (CENPC-CT 3A679-681), and CENPC-CT, in which five residues were deleted (CENPC-CT ∆678-683), are shown. C) EMSA results of the binding affinities of CENPC-CT WT, CENPC-CT 3A679-681, and CENPC-CT ∆678-683 to the CENP-A nucleosome. D) Stable expression of GFP-fused CENPC-CT and its mutants (shown in (B)) in CENP-C knockout chicken DT40 cells. α-Tubulin (Tub) was probed as a loading control. Parental CENP-C knockout cells (parental) were also examined. E) Localization analysis of GFP-fused CENPC-CT WT, CENPC-CT 3A679-681, and CENPC-CT ∆678-683 (green) on the mitotic chromosomes in chicken DT40 cells. CENP-T (red) was used as a centromere marker. DNA was stained using 4',6-diamidino-2-phenylindole (DAPI; blue). Scale bar indicates 10 μm. F) Localization of CENP-C in CENP-A knockout DT40 cells stably expressing GFP-fused gCENP-A WT, RG-AA, or ∆RG. Scale bar indicates 10 μm. CENP-C was stained by an anti-CENP-C antibody (red) and DNA was stained by DAPI (blue). CENP-C signals on kinetochores in mitotic cells were quantified in each cell. Bar graph indicates mean ± SD (n = 7; ****, P < 0.0001, unpaired t test, two tailed).
Both the PB1 and UBA domains are needed for p62 to form cytoplasmic bodies.Figure 2.(A) The indicated deletion constructs were fused COOH terminal to GFP and expressed in NIH3T3 fibroblasts. Asterisks indicate point mutations in the PB1 (D69A) and UBA (I431A) domains, respectively. (B) S-GFP-p62 cells were transiently transfected with the indicated myc-tagged p62 constructs, fixed, and stained for the myc tag. Bars, 20 μm.
The FYVE domain protein blocks LC3 lipidation of the 25K membrane pellet fraction.Figure 1--figure suppleme
Figure 1Schematic of the mTORC1 and mTORC2 complexes and inhibitors. Images of mESCs treated with control (Ctrl, dimethyl sulfoxide (DMSO)), INK (INK128, 200 nM) for three days and released for three days in 2iL. Scale bar, 400 µm. Relative proliferation of mESCs as in (B). Data are the mean values ± SD with the indicated significance (P value was calculated using two-tailed unpaired Student's t test, ***P < 0.001, n.s., not significant, also hereafter in similar experiments). n = 3 biological replicates. Western blotting for mTORC1 and mTORC2 activity in mESCs transduced with Rptor and Rictor shRNAs. Images of mESCs transduced with Rptor and Rictor shRNAs in 2iL. Scale bar, 400 µm.   Relative proliferation of mESCs as in (E) (mean values ± SD, P value was calculated using two-tailed unpaired Student's t test, *P < 0.05, also hereafter in similar experiments). n = 3 biological replicates.   Western blotting for mTORC1 and mTORC2 activity in Rptor WT (AAV-empty) and KO (AAV-Cre) mESCs. Samples were collected after AAV transduction for four days. Images of Rptor WT and KO mESCs in 2iL. Scale bar, 400 µm. Western blotting for RPTOR and the indicated pluripotency proteins in mESCs as in (H) (left), and quantification of pluripotency proteins (right) (mean values ± SD). n = 3 biological replicates. Images of mESCs transduced with Rptor siRNA on different days in 2iL. siNC, transduced with a negative control; day 0 was the day of siRNA transfection, also hereafter in similar experiments. Scale bar, 400 µm. Relative proliferation of mESCs as in (J) (mean values ± SD). n = 3 biological replicates. Images of EBs on different days after INK128 release for three days. Scale bar, 400 µm. Relative number of beating EBs on D12 as in (L) (mean values ± SD). n = 3 biological replicates.
Figure 4A. Schematic illustration of psicheck2-based luciferase reporter constructs used for examining the effect of miR-586 on the 3' UTR of Fbxw7. Mutated bases are in red color in open box. B. miR-586 mimics or negative control mimics (NC mimics) were transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test). C. miR-586 inhibitor or negative control inhibitor (NC inhibitor) was transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test). D. HeLa cells were transfected with miR-586 mimics or inhibitors as indicated. Forty-eight hours after transfection, cell lysates were analyzed by Western blotting. E. HeLa cells were transfected with miR-586 mimics or inhibitors as indicated. Forty-eight hours after transfection, total RNA was extracted and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test). F. The copy numbers of miR-586 and lncRNA-MIF transcript per cell in the indicated cells were quantified using a quantitative real-time RT-PCR assay. Data shown are mean ± SD (n = 3). G. HeLa cells were infected with lentiviruses expressing control shRNA or lncRNA-MIF shRNA. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. HeLa cells were infected with lentiviruses expressing lncRNA-MIF or control RNA. Forty-eight hours after infection, total RNA was analyzed by real-time RT-PCR. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test). H. HeLa cells expressing lncRNA-MIF or control RNA were transfected with miR-586 mimics or NC mimics. Twenty-four hours after transfection, cell lysates were analyzed by Western blotting. I. HeLa cells expressing lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor. Twenty-four hours after transfection, cell lysates were analyzed by Western blotting. J. Mutant bases (in red color) in nonsense mutant lncRNA-MIF (negative control, NC) and in miR-586 binding defective lncRNA-MIF are shown in boxes. K. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, lncRNA-MIF harboring nonsense mutation or miR-586 binding defective lncRNA-MIF. Forty-eight hours after transfection, cell lysates were analyzed by Western blotting.
a) Immunoblot for LC3-II in control mouse fibroblasts (Ctr) or those knocked down for RARα (RARα(−)) maintained in the presence or absence of serum for the indicated times.figf2(a) Immunoblot for LC3-II in control mouse fibroblasts (Ctr) or those knocked down for RARα (RARα(−)) maintained in the presence or absence of serum for the indicated times. Where indicated, protease inhibitors (PI) against lysosomal proteolysis were added. Actin is shown as a loading control. 'I' and 'II' designate different forms of LC3, as described in the text. (b) Amounts of LC3-II determined by densitometric quantification of immunoblots. Values are expressed as fold change in value relative to serum-supplemented control cells; n = 4. (c) Ratio of the amounts of LC3-II in cells treated with protease inhibitors compared to untreated cells (None). Values are expressed as fold change in value relative to untreated cells; n = 4. (d,e) Autophagic flux in the same cells as in c expressing mCherry-GFP-LC3-II and maintained in the presence or absence of serum. Shown in d are representative merged channel images; scale bars, 2 μm. Arrows indicate autolysosomes (red). Shown in e are the quantification of the number of autophagosomes (mCherry and GFP positive) per cell (left) and percentage of autolysosomes (mCherry positive and GFP negative; right) in >50 cells in at least four different fields. AV, autophagic vacuoles. (d,e) Autophagic flux in the same cells as in c expressing mCherry-GFP-LC3-II and maintained in the presence or absence of serum. Shown in d are representative merged channel images; scale bars, 2 μm. Arrows indicate autolysosomes (red). Shown in e are the quantification of the number of autophagosomes (mCherry and GFP positive) per cell (left) and percentage of autolysosomes (mCherry positive and GFP negative; right) in >50 cells in at least four different fields. AV, autophagic vacuoles. (f) Control and RARα(-) cells were transfected with the KFERQ-mCherry1 photoactivatable reporter, and after photoactivation they were maintained in medium with or without serum. Left, representative images. Graph shows quantification of the number of puncta per cell in >50 cells using the CMA reporter in at least four different fields. Nuclei are labeled with DAPI. Scale bars, 10 μm. In b, c, e and f, all values are mean ± s.e.m. Differences with control (marked with asterisk) or with serum-supplemented cells (marked with §) are significant for P 0.05. Full-field fluorescence images and full-length blots are shown in Supplementary Figures 2 and 21, respectively.
Figure 3A) Structure of BID (PDB:2BID) depicting residues G94, D95, and M97-D98 localized at the BH3 domain in light orange, and K157/K158 at the α-helix 6 in blue. B) Cell death induced by tBID-GFP and its variants on HCT AKO, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to three independent experiments, with n> 30 cells per condition per experiment. *** p<0,001 and * p<0,05 with respect to tBID-GFP WT condition. C) Structure of BID (PDB:2BID) depicting cancer-related residues localized at α-helix 6 in blue. D) Effect of tBID wt and α6-mutants overexpression on HCT AKO cells, measured as DRAQ7+/GFP+ cells and normalized to tBID wt condition. n=3 independent experiments, ** p<0,025 and * p<0,05 with respect to tBID-GFP wt condition. E) Effect of tBID wt and α6-mutants overexpression on MEF BID KO cells, measured as DRAQ7+/GFP+ cells and normalized to tBID wt condition. Effect of GFP overexpression on BID KO cells relative to tBID wt was assessed by pyknotic nuclei formation quantification. n=3 independent experiments *** p<0,001 with respect to tBID-GFP wt condition.
Figure 1(A) NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 strain at MOI 10 and 0.01, respectively. At the indicated time points, the culture supernatants were removed and the cell lysates were prepared for Western blot analysis. Relative protein levels were determined by normalizing against β-actin and compared to mock-infected cells (B) NSC-34 and SH-SY5Y cells were transfected with various concentrations of siPRPH or siNTC. The efficiency of siRNA knockdown at 48h.p.t. was validated by Western blot while cytotoxicity was determined by AlamarBlue assay. siPRPH- or siNTC-transfected cells were infected with EV-A71 S41, and the viral titers were determined at 48h.p.i. (C) PRPH was overexpressed in NSC-34 cells upon transfection with pCMV6-entry vector containing PRPH gene At 48h.p.t., cells were infected with EV-A71 S41 at MOI 5 and the viral titers were determined at 48h.p.i. (D) SH-SY5Y cells were transfected with various concentrations of siPRPH or siNTC. At 48 h.p.t. cells were infected with CA16 or EV-A71 C2 at MOI 0.01. Viral titers were determined 48 h.p.i by plaque assay.
ASGP-R labeling in FP+ and endosomes.Figure 7ASGP-R labeling in FP+ and endosomes. Cells exposed for 40 min to 5-nm gold tracer. Section immunolabeled for ASGP-R with 10-nm gold. (a) Low ASGP-R labeling in tracer-carrying endocytic vesicles (arrowheads), adjacent to (top) or fusing with (bottom) an AVi/d. (b) ASGP-R labeling at the plasma membrane and in endosomal elements near the cell surface (ex, extra cellular space). Vesicles of the MVE (asterisk) and VE (arrowhead) type, containing endocytosed gold, are present in the same area. (c) As b. A VE-like vesicle with endocytosed gold and low ASGP-R labeling (arrowhead) is shown connected to an ASGP-R rich element (arrow), possibly a recycling endosome. Bars, 200 nm.
Figure 2A Confocal images of primary hepatocytes grown under indicated conditions and treatments after ribopuromycylation assay stained with anti-puromycin antibody. Scale bar, 10 μm. B Quantification of fluorescence intensity per cell of experiment shown in (A). AU, arbitrary units. (n= 90-110 cells per treatment isolated from 4 mice). Data information data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. C Quantification of the number of puromycin granules under indicated conditions (n ≥ 50 cells isolated from 4 mice). Data information: data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001; **, P≤0.01 (Student's t-test). D Confocal images of primary hepatocytes stained with anti-puromycin and anti-CLUH antibodies. The cells from which the enlarged areas (400 μm2) have been magnified are shown in Appendix Figure S1B for each individual channel. Scale bar, 4 μm. Cells analyzed were isolated from 4 different mice with similar results. Arrows point to colocalizing particles. E Manders' colocalization coefficient between puromycin and CLUH from experiment shown in (D) (n ≥ 50 cells isolated from 4 mice). Data information: data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. (E) ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test). F Quantification of puromycin granules containing CLUH signal (n ≥ 80 cells isolated from 4 mice). Data information: data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001; **, P≤0.01 (Student's t-test).
Overview of enzyme-metabolite interactions detected with NMR A, Number of interactions detected per metabolite.Figure 3Number of interactions detected per metabolite. Metabolites are grouped according to biological pathways; the height of the bar indicates the total number of interactions. Recovery of interactions reported in EcoCyc database. Metabolites are grouped according to biological pathways, the total height of the bar indicates the number of known interactions that could have been detected, the height of the colored bar indicates the number of actually recovered interactions. Recovery of catalytic (metabolite is substrate or product of the enzyme) and regulatory interactions is shown. Distribution of known regulatory and newly predicted interactions in E. coli central metabolism. Enzymes included in this study are depicted in bold. Grey and white circles indicate how many known interactions were recovered or not recovered, respectively. Newly detected interactions are depicted using circles that are color coded according to the origin of the respective metabolite.
Figure 1A, Domain organization of CNNM magnesium transporters and PRL phosphatases. B, ITC experiment shows the CBS-pair domain of CNNM3 binds PRL2 with a nanomolar Kd and one-to-one stoichiometry. C, Sequence alignment of the extended loop in the CBS-pair domains of CNNM proteins. The loop is a unique to the four human CNNM family members compared to related CBS-pair domain proteins: MAM3, CorC efflux protein from Haemophilus influezae, and the MgtE Mg2+ transporter from Enterococcus faecalis. Loop residues unique to CNNM proteins are highlighted in yellow. D, Structure of CBS-pair domain of CNNM2 (PDB 4IY0) with the residues DPFYE indicated. E, Schematic of catalytic cycle of PRL phosphatases. Catalysis proceeds through a long-lived phosphocysteine intermediate. Turnover of the intermediate is the rate-limiting step. F, ITC data of CBS-pair domain mutants binding to PRL2 identifies aspartic acid 426 as essential for binding. G, Kinetics of hydrolysis of a synthetic substrate by PRL2 shows burst kinetics. Addition of the wild-type CBS-pair domain but not the D426A mutant inhibits phosphatase activity.
Protein import machinery.Figure 6(A-D) Components of the mitochondrial protein import machinery and selected proteins related to this process of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored and dark-grey bars). A and B, components of the translocase of the outer membrane (TOM) machinery (TOM avg.: average of TOMM40, TOMM22, TOMM5, TOMM6, TOMM7); C, components of the trans­locase of the inner membrane (TIM) machinery (TIM22 avg.: TIMM22, AGK, TIMM10B; TIM23 avg.: TIMM23, TIMM17A, TIMM17B); D, components of the TIM23-associated TIMM21 assembly machinery (left panel) and its association with prohibitin (PHB) and components of the mitochondrial quality control system (right panel). Plots and gene names are in corresponding colors, except for TIMM44 and TIMM50 (no migration profiles shown). In (A-C) data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test).
Figure 7A) HIF1α and BNIP3 protein expression was measured in gastrocnemius muscle from control and Mfn2KO young mice untreated or treated with NAC (n=3-5 mice per group). Data were normalized by tubulin as a loading control and expressed as a fold change compared to young control mice. B) Respiration was evaluated in control and Mfn2KD C2C12 myotubes untreated or treated with NAC. ATP-coupled respiration (left panel) and proton leak (right panel) were calculated (n=5 independent experiments). Data were expressed as a fold change compared to control C2C12 myotubes. C) Expression of LC3II and Parkin was evaluated in mitochondrial enriched fractions from control and Mfn2KD C2C12 myotubes untreated or treated with NAC (n=5 independent experiments). Data were normalized by TIM44 as a loading control and expressed as a fold change compared to control C2C12 myotubes. D) Expression of LC3II and Parkin was evaluated in mitochondrial enriched fractions in gastrocnemius muscle from control and Mfn2KO mice untreated or treated with NAC (n=5 mice per group). Data were normalized by TIM44 as a loading control and expressed as a fold change compared to young control mice. E) Representative TEM images of control and Mfn2KD C2C12 myotubes treated with NAC showing increased number of autophagosomes and mitochondrial alterations (n=3 independent experiments) (Scale bar: 2 µm). F) Proposed model for the role of Mfn2 during aging in skeletal muscle. Data represent mean ± SEM. #p<0.05 Mfn2KO vs. control mice or Mfn2KD vs. control cells, *p<0.05 old vs. young mice or treated vs. untreated cells.