EMBO/sd-character-level-ner huggingface dataset:
Figure 3A Electron micrographs of METTL3-knockdown SMMC-7721 cells under hypoxia for 48 h. Protein levels of LC3 I/II in METTL3-knockdown SMMC-7721 (B), Bel-7402 (C) cells under hypoxia condition for 48h. Protein levels of LC3 I/II in HepG-2 (D) cells under hypoxia condition for 48h. E Protein level of LC3 I/II in HepG-2 and sorafenib-resistant HepG-2 cells under hypoxia condition for 48h. F-H Overexpression of shRNA-resistant wild type METTL3 but not catalytic mutant METTL3 rescue autophagy phenotype in METTL3-knockdown SMMC-7721 cells (F), Bel-7402 cells (G) and sorafenib-resistant HepG2 cells (H) by representative immunostaining images of LC3 under hypoxia condition for 48h. Scale bar, 200μm. Quantification of the numbers of GFP-LC3 puncta/cell by Imaris in F-1, G-1 and H-1, respectively. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments. I-K Protein levels of LC3 I/II in METTL3-knockdown SMMC-7721 cells (I), Bel-7402 cells (J) and HepG-2 cells (K) treated with or without 3-MA under hypoxia condition for 24h. L-N The IC50 of sorafenib in SMMC-7721 cells (L), Bel-7402 cells (M) and HepG-2 cells (N) treated with or without 3-MA for 24h under hypoxia condition (1%O2). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.
Figure 2Figure 2. Model size and performanceClassification performance of varying numbers of top-ranking genes in RF models built and cross-validated on the training set. Displayed are AUC values from 25 reiterative RF models with boxplot overlay.
The structure of the CSS as well as a summary of the subcellular localization, ability to translocate during mitophagy and number of basic amino acids in the CSS are shown for each protein.f6The structure of the CSS as well as a summary of the subcellular localization, ability to translocate during mitophagy and number of basic amino acids in the CSS are shown for each protein.
Figure 7(A-C) One cohort of mice was assessed with the social approach test at young (A) and old (B) age and another cohort of mice at middle age (C). Mice were placed individually into the empty center chamber of a three-chamber apparatus. One side-chamber contained an empty cup, and the other an identical cup with an unfamiliar live mouse in it. Close contact with each cup was monitored for 10 min. n = 12 (A, B) or 17 (C) NTG; 13 (A) or 10 (B) TRE-hTau-A152T(L1); 12 (A), 9 (B), or 12 (C) CamKII-tTA; 13 (C) hTau-WT(L32); and 14 (A), 12 (B), or 10 (C) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by one-tailed paired t test with Holm adjustment. Values are means ± SEM.
Tissue regularity is a function of cell proliferation.Figure 2Egg chamber elongation is unaffected by reduced cell proliferation. String-shRNA expressed for 24 hours alters epithelial shape and cell number (A Chambers shown in (A) have approximately equal widths, with the cross-sectional area corresponding to developmental stage 6. Scale bars = 20 μm. Number of chambers analyzed: w1118 1.0-1.2 n=24,1.2-1.4 n=14,1.4-1.6 n=18 String-shRNA 1.0-1.2 n= 11 ,1.2-1.4 n=10,1.4-1.6 n=15. Bars represent mean and standard deviation Egg chamber elongation is unaffected by reduced cell proliferation. String-shRNA expressed for 24 hours alters epithelial shape and cell numbe but not the relationship between egg chamber aspect ratio and cross-sectional area, which is quantified in (B) Number of chambers analyzed: w1118 1.0-1.2 n=24,1.2-1.4 n=14,1.4-1.6 n=18 String-shRNA 1.0-1.2 n= 11 ,1.2-1.4 n=10,1.4-1.6 n=15. Bars represent mean and standard deviation C, D) Cell proliferation is necessary for normal tissue morphology Follicle cell shape regularity (C) and polygonality (D) measurements in control and String-shRNA egg chambers of different aspect ratios. These measurements were derived from at least three egg chambers from at least three flies per AR range. Bars represent mean and standard deviation. Statistical significance for cell side distributions was calculated using a chi-squared test. Number of cells used for analysis: w1118 1.0-1.2 n= 23, 1.2-1.4 n=114, 1.4-1.6 n=160; String-shRNA 1.0-1.2, n=6, 1.2-1.4 n=19, 1.4-1.6 n=17
Loss of IL-2 priming correlates with reduced gene expression of proximal genes A.Figure 5Principle component analysis (PCA) of TB IL-2, TB IL-2nil, TB+ IL-2 and TB+ IL-2nil from 3 replicates. Hierarchical clustering of RNA-Seq data from 3 replicates as described in (A). MA plot showing the genes differentially regulated between TB IL-2nil and TB IL-2. The number of genes with a fold change >3 and adj p-value <0.05 are highlighted. P-values were calculated from the RNA-seq data using Limma with Benjamini-Hochberg correction for multiple testing. log2 fold change in gene expression in TB IL-2nil compared to TB IL-2 for the nearest gene to the DHSs ranked according to the fold change in DNase-Seq tag count for TB IL-2/TB IL-2nil. The DNase-Seq tag density plots are taken from Figure 2A for TB IL-2, TB IL-2nil and TN. UCSC genome browser tracks showing DNase-seq and RNA-seq at the Socs1 (E) and Sell loci (F).
Fig.Figure 7(A) The proteolytically dead mutant YqgP.S288A behanes as a pseudoprotease and acts as a substrate adaptor of FtsH. Pattern of C-terminally truncated forms of endogenous MgtE formed in vivo in the presence of ectopically expressed active YqgP (strain BS72, Table EV2) or its inactive S288A mutant (BS73, Table EV2) in rich LB medium in the presence or absence of endogenous FtsH. Black arrow denotes the full-length MgtE substrate, and red arrows denote its cleavage products generated by YqgP. While the presence of the faster migrating YqgP-dependent products (full red arrows) do not depend on the presence of FtsH, the slower migrating product formed in the presence of YqgP.S288A (open red arrow) does, indicating that FtsH protease is responsible for the alternative cleavage of MgtE. The same arrow symbolics are valid throughout this figure. (B) Mapping of the boundary of the FtsH generated proteolytic product of MgtE. Immunoblot comparing the SDS PAGE mobility of the N-terminal fragment of endogenous MgtE generated by FtsH in the presence of the pseudoprotease version of YqgP (catalytically dead YqgP.S288A, strains BTM78, BTM502, Table EV2) to the mobilities of in vitro translated reference fragments corresponding to MgtE 1-340, -355 and -370 shows that YqgP.S288A acts as an FtsH adaptor, and that FtsH processing of MgtE in the presence of YqgP.S288A stops at around amino acid 355, which is near or at the cytoplasmic end of TMH 3 of MgtE. (C) Schematic depiction of the regions in MgtE defining its remaining cleavage products formed by FtsH and YqgP. (D) In vivo cleavage of endogenous MgtE in the presence of ectopically expressed full-length YqgP.S288A (strain BS73), YqgPΔNTD.S288A (BS185, Table EV2) or YqgPΔCTD.S288A (BS186, Table EV2), in rich LB medium shows that YqgPNTD is necessary for the FtsH-dependent processing of MgtE. (E) Analysis of the in vivo kinetics of formation and fate of MgtE cleavage products. B. subtilis strains expressing YqgP or its S288A mutant (strains BTM78; 501 and 502, Table EV2) in the presence or absence of endogenous FtsH protease (strains BTM795, BTM796 and BTM797, Table EV2) were cultured in LB medium and at early exponential phase, after having been expressing YqgP variants for 30 min, were either left grown untreated (as a control with ongoing translation) or treated with 20 μg/mL tetracycline to stop proteosynthesis (translation shut-off). At given time intervals afterwards, all cultures were analysed by α-MgtE western blotting with near infrared detection. Equal cell number (judged by OD600) was loaded into each lane. Black arrow marks full-length MgtE, red full arrows mark YqgP-dependent and red open arrows mark YqgP.S288A-dependent cleavage product of MgtE.
Fig.Figure 5(A) Detection (left panel) and quantification (right panel) of steady-state conversion of endogenous MgtE by YqgP (strain BS72, Table EV2) grown in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (reference conditions) with the additions of 10 µM or 100 μM of a given divalent cation salt solution (MnCl2, ZnCl2, CoCl2, or NiCl2), or 50 µM and 500 μM for CaCl2. Western blots were quantified by near-infrared fluorescence detection (left), quantified by densitometry and displayed as relative specific activity (graph on the right), which is substrate conversion normalized to enzyme expression level. Black arrow denotes full-length substrate (MgtE), and red arrow denotes the N-terminal cleavage product(s) generated by YqgP. All bands originate from the same western blot and identical treatment series. (B) Isothermal titration calorimetry of purified recombinant YqgPNTD and selected divalent cations. (C) Cation toxicity assays and their relationship to YqgP activity. Wild type B. subtilis (BTM2, Table EV2), its variant lacking YqgP (ΔyqgP, BTM78, Table EV2), and the rescue strain ectopically expressing YqgP (BTM501, Table EV2) were cultivated in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (i.e. reference conditions), and in mid-exponential phase were stressed by the addition of either 70 µM MnCl2, 500 µM ZnCl2, 25 µM CoSO4 or 400 µM NiCl2 (pale blue area). YqgP activity specifically improved cell fitness during manganese and zinc stress, while it had no effect on growth of cells cultivated in the presence of high cobalt and nickel concentrations. Representative experiments of 2-3 independent replicates are shown.
gba−/− Mitochondria Have a Reduced Respiratory Chain Activity.Figure 4(A) Oxygen consumption rates of gba+/+, gba+/−, and gba−/− mixed midbrain cultures. Basal oxygen consumption was measured over 3 min. The maximal (uncoupled) rate was measured via the addition of FCCP and the nonmitochondrial oxygen consumption analyzed via the addition of antimycin A (n = 3, three runs/experiment). (B) Complex I-NADH: Ubiquinone reductase activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3). (C) Complex II-III: Succinate dehydeogenase, cytochome c reductase activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3). (D) Complex IV activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3). (E) Neurons were treated with 3 nM decylTPP and MitoQ10 for 48 hr and stained with TMRM and fluorescence intensity analyzed via confocal microscopy (n = 3, >16 cells/experiment). (F) gba+/+and gba−/− cells treated with 3 nM decylTPP and MitoQ10 for 48 hr were subjected to immunoblotting using the indicted antibodies. All data in this figure represent the mean ± SEM, *p < 0.05.
Fig.Figure 1A. Overview of transcriptomic (top) and proteomic (bottom) analyses. B. Over-expression analysis of enriched KEGG pathways of significantly differentially expressed genes between low STAT3 versus high STAT3 groups in TCGA PRAD.
Figure 9fEPSPs in CA3 stratum lucidum were induced by stimulation of mossy fibers in acute hippocampal slices. (A) Representative fEPSP traces from slices of 4-8-month-old mice. (B, C) Input/output curves reflecting the change in the fEPSP slope as a function of the fiber volley amplitude from slices obtained at 4-8 (B) or 20 (C) months of age. (D, E) Short-term plasticity assessed by measuring the PPR (second pulse/first pulse) with 50-msec interpulse interval at 4-8 (D) or 20 (E) months of age. (F) Mossy fiber LTP at 20 months of age. n (mice/slices) = 5-9/8-21 (NTG), 2-5/5-12 (CaMKII-tTA), 3-6/8-14 (TRE-hTau), 3-6/8-14 (hTau-WT), and 5-6/8-14 (hTau-A152T). *P < 0.05, **P < 0.01, ***P < 0.001 versus NTG by linear regression analysis with Holm adjustment (B, C) or by one-way ANOVA and Dunnett's test (D, E). No significant difference was detected by two-way ANOVA in (F). Values are means ± SEM.
A) Redistribution of CMA-active lysosomes induced by overexpression of LAMP-2A.Figure 3(A) Redistribution of CMA-active lysosomes induced by overexpression of LAMP-2A. PC12 cells were transfected with PCDNA4-LAMP-2A for72 h. Lysosomes were labeled with immunofluorescence of Cathepsin D (green) and the increased perineuclear localization of CMA-active lysosomes was observed. N: nucleus. Thin arrows point to lysosomes. The scale bar = 10 µm. (B) The efficiency of knockdown and overexpression of LAPM-2A. PC12 cells were transiently cotransfected with LAMP-2A plasmid or LAMP-2A siRNA for 48 h. Lysates were subjected to Western blot analysis with anti-human LAMP-2A. Values are the mean±SE of three independent experiments. **P<0.01 (compared with control); ##P<0.01 (compared with Htt-552-100Q with control). (C) and (D) Effects of knockdown and overexpression of LAMP-2A on Htt-552 levels. PC12 cells were transiently transfected with LAMP-2A plasmid, vector control or LAMP-2A siRNA for 72 h and Htt-552 for 48 h. Lysates were subjected to Western blot analysis with anti-Htt 2166, anti-human LAMP-2A antibodies. Values are the mean±SE of three independent experiments. *P<0.05 (compared with Htt-552-18Q without treatment); #P<0.05 (compared with Htt-552-100Q without treatment). (C) and (D) Effects of knockdown and overexpression of LAMP-2A on Htt-552 levels. PC12 cells were transiently transfected with LAMP-2A plasmid, vector control or LAMP-2A siRNA for 72 h and Htt-552 for 48 h. Lysates were subjected to Western blot analysis with anti-Htt 2166, anti-human LAMP-2A antibodies. Values are the mean±SE of three independent experiments. *P<0.05 (compared with Htt-552-18Q without treatment); #P<0.05 (compared with Htt-552-100Q without treatment). (E) Redistribution of CMA-active lysosomes induced by overexpression of Hsc70. PC12 cells were transfected with Hsc70 plasmid for 48 h. Lysosomes were labeled with immunofluorescence for Cathepsin D (green) in cultured PC12 cells and the re-location of CMA-active lysosomes toward the perinuclear regions was observed. N: nucleus. Thin arrows point to lysosomes. The scale bar represents 10 µm (F) The efficiency of knockdown and everexpressiom of Hsc70. PC12 cells were transiently transfected with Hsc70 plasmid and Hsc70 siRNA for 48 h. Lysates were subjected to Western blot analysis with anti-Hsc70 antibody. Values are the mean±SE of three independent experiments (**P<0.01 vs control). (G) Effects of knockdown and overexpression of Hsc70 on Htt-552 levels. PC12 cells were transiently transfected with human Hsc70 plasmid, vector control or Hsc70 siRNA for 72 h and Htt-552 for 48 h. Lysates were subjected to Western blot analysis with anti-Htt 2166, anti-Hsc70 antibodies. Values are the mean±SE of three independent experiments. **P<0.01 (compared with Htt-552-18Q without treatment); ##P<0.01 (compared with Htt-552-100Q without treatment).
Figure 3Representative Coomassie-stained SDS-PAGE gel showing the isolation of SDS insoluble aggregates from mouse brain lysates. TH = total homogenate, SUP = supernatant, W1 = SDS-soluble fraction, Pellet = formic-acid soluble fraction (see Figure EV4A). Quantification of the yield of SDS-insoluble aggregates from young and old brain lysates was based on densitometry analysis of Coomassie-stained gel bands obtained from different animals, n=5 per age group (Figure EV4B); ** p < 0.01, unpaired t-test. In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range. Proteins enriched in aggregates show a predicted higher molecular chaperone requirement for folding (top vs. bottom 20% p=0.0055, Kolmogorov-Smirnov test), and are richer in intrinsically disordered regions (s2D-derived scores, top vs. bottom 20% p=0.0019, Kolmogorov-Smirnov test). Violin plots: the solid line shows the median, the dotted lines the interquartile ranges. The same result was obtained with cleverSuite-derived scores (top vs. bottom 20% p=0.0201, Kolmogorov-Smirnov test) (Figure EV4C). ** p < 0.01, Kolmogorov-Smirnov test. Volcano plot based on protein quantification by label-free mass spectrometry depicting the enrichment of specific proteins in protein aggregates. The x-axis indicates the log2 ratio between protein abundance in aggregates (Pellet) and starting total homogenate (TH). The horizontal dashed line indicates a p value cut-off of 0.05 and vertical lines a log2 fold change cut off of ± 0.5. Selected proteins are highlighted as colored dots as indicated in the figure legend. Protein quantification was based on samples obtained from 3 independent isolations. Double-labelling of telencephalic sections of N. furzeri with anti-RPS6 (green) as ribosomal marker and Proteostat as a marker for aggregated proteins (red). Nuclear counterstaining was performed with DAPI (blue). Scale bar = 10 μm.
is preferentially expressed in Treg subsets.Figure 1A. (Top) USP44 transcript levels across CD4+ T cell populations. Suspension of the lymph node and spleen cells of 6-8 week old female C57BL/6 mice (n= 5 experiments) were obtained, and naïve (CD62Lhigh/CD25-) CD4+ T cells and nTregs (CD4+/CD25high) were obtained by FACS. The indicated effector T cell populations were generated by activating 1x10^6 naïve CD4+ T precursors with anti-CD3 and anti-CD28 antibodies (1 and 4 ug/ml, respectively) for 4 days under the indicated in vitro skewing conditions (described in the Methods section). After harvesting RNA by Trizol reagent and generating cDNA, the Usp44 mRNA levels expressed by the resulting Teff and iTreg, as well as freshly isolated nTreg cells were determined by qRT-PCR. (Bottom) Suspension of lymph node and spleen cells of 6-8 week old female C57BL/6 mice were obtained, and naïve (CD62LhighCD25-) CD4+ T cells and nTregs (CD4+/CD25high) were sorted by FACS. iTregs were generated by activating 1x10^6 naïve CD4+ T cells with anti-CD3 and anti-CD28 antibodies (1 and 4 ug/ml, respectively) for 4 days in the presence of IL-2 (100u/ml) and TGF-β (5ng/ml) before the cells were harvested for SDS-PAGE. Panels A and B depict the mean mRNA expression normalized to the housekeeping gene GAPDH. B. Naïve murine CD4+ T cells were obtained and differentiated into the iTreg lineage as in A. Expression levels of Usp44 and Foxp3 mRNA were assessed daily over time by qRT-PCR. C. Expression of USP44 and FOXP3 protein by T cells during in vitro differentiation was also measured. After 1-4 days of iTreg skewing, cell lysates were resolved by SDS PAGE following by immunoblotting with the indicated antibodies. D. Murine iTregs generated in vitro as above were activated in the presence of recombinant TNFα, IL-6, or IL-1β (each at a 10 ng/ml concentration), or LPS (1µg/ml) for 24 hours. Treated Tregs were then harvested and USP44 protein levels were determined as above.
Cvt complexes in TVY1 (pep4) cells under nitrogen starvation conditions.Figure 3Cvt complexes in TVY1 (pep4) cells under nitrogen starvation conditions. Logarithmically growing cells in YEPD were transferred to nitrogen starvation medium (SD[-N]) at 30°C for 30 min (A and B) or 60 min (C and D) and prepared for electron microscopy as described in Fig. 1. (A) Freeze-substitution fixation image showing a Cvt complex in an autophagosome (marked by an arrow) in the cytosol. (B) Freeze-substitution fixation image showing a Cvt complex in an autophagic body in the vacuole. (C) Immunostaining of a Cvt complex in an autophagic body in the vacuole. (D) Immunostaining of Cvt vesicles (marked with arrows) in the vacuole. AB, Autophagic body; AP, autophagosome; V, vacuole.
Figure 1A The sequences of the isolated VHH antibodies (49 anti-RBD and 10 anti-S1ΔRBD nanobodies) were aligned using Clustal Omega (Sievers et al., 2011). The circular phylogram was reconstructed with Dendroscope (Huson and Scornavacca, 2012). Where applicable, sequence classes are indicated. The anti-RBD nanobodies are colored according to the two RBD epitopes identified in Fig 5 (with epitope 1 in green, epitope 2 in red, and asterisks marking nanobodies included in Fig 5). Anti-S1ΔRBD nanobodies are colored in magenta. VHH antibodies that did not neutralize SARS-CoV-2 are shown in parentheses. B Vero E6 cells were inoculated with SARS-CoV-2, fixed with paraformaldehyde after two days, stained with 30 nM of the indicated Alexa Fluor 488 (AF488)-labeled anti-RBD VHH antibodies, and imaged by confocal laser scanning microscopy (CLSM). C Immunofluorescence (IF) staining as in B, but with the indicated AF568-labeled anti-S1ΔRBD VHH antibodies.
Figure 1A Dose-response binary food choice assay of saponin (n ≥ 4). B Dose response curve of tip recordings using the indicated concentrations of saponin on S6 sensilla (n ≥ 8). C Sample traces of saponin-induced action potentials in (B). D Dose response curve for sugar inhibition using the indicated concentrations of saponin with 100 mM sucrose on L4 sensilla (n ≥ 8). E Sample traces from (D). The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis as a post hoc test to compare two sets of data (**P < 0.01). The error bars represent SEMs.
Preventive effect of R-III on CCl4-induced liver fibrosis.Figure 8Liver sections from control and CCl4- and CCl4/R-III-treated mice were stained with Sirius red. Scale bar, 200 μm. Hydroxyproline content in the livers (μg/g liver). *P = 0.034, two-sample t-test (n = 10) (CCl4 + R-III compared to CCl4-treated mice).
Figure 6A. qRT-PCR analysis of Fam134b isoforms in mock, TFEB S142A:S211A and TFE3 S246A:S312A over-expressing U2OS cells. Values were normalized to Hprt gene and expressed as fold change values relative to mock. Mean +/- standard error of the mean (sem) of N=3 biological replicates. Student's un-paired T-test *p<0.05; NS not significant. B. Western blot analysis of Fam134b isoforms in mock, TFEB S142A:S211A and TFE3 S246A:S312A over-expressing U2OS cells showing induction of FAM134B-2, but not of FAM134B-1 isoform by TFEB/TFE3. Filamin A was used as loading control. Blot is representative of N=3 independent experiments. C,D. Co-immunofluorescence staining of ER (CLIMP-63, green) and lysosomes (TMEM192-HA, red) in control and Sh-FAM134B U2OS cells over-expressing TFEB S142A:S211A-GFP (purple). BaFA1 was used at 100 nM for 4h. Scale bar 15 μm and 2 μm (higher magnification boxes) (C). In (D), Quantification of CLIMP63 fluorescence in TMEM192-HA decorated lysosomes. Mean +/- standard error of the mean (sem) of N=3 biological replicates. N= 21 (vehicle ctrl), N=33 (TFEB S142A:S211A-GFP ctrl), N=21 (vehicle shFAM134B), N= 30 (TFEB S142A:S211A-GFP shFAM134B) cells were analysed. Student's un-paired T-test ***p<0.0005; NS not significant. E,F. Co-immunofluorescence staining of ER (CLIMP-63, green) and lysosomes (Lamp1, red) in WT and Fam134bKO MEF cells over-expressing TFEB S142A:S211A-GFP (purple). BaFA1 was used at 100 nM for 4h. Scale bar 15 μm and 2 μm (higher magnification boxes) (E). In (F), Quantification of CLIMP63 fluorescence in Lamp1 decorated lysosomes. Mean +/- standard error of the mean (sem) of N=3 biological replicates. N= 20 (vehicle ctrl), N=22 (TFEB S142A:S211A-GFP ctrl), N=21 (vehicle Fam134bKO), N= 20 (TFEB S142A:S211A-GFP Fam134bKO) cells were analysed. Student's un-paired T-test ***p<0.0005; NS not significant.