EMBO/sd-character-level-ner huggingface dataset:
M45 interacts with the IKK complex.Figure 3(A) 293A cells were transfected with plasmids expressing HA-tagged M45 or an unrelated MCMV control protein (m143) and Flag-tagged IKK subunits. Lysates were subjected to immunoprecipitation (IP) using an anti-HA antibody. Immunoprecipitates were analyzed by immunoblotting (IB) using anti-Flag antibody. Whole cell lysates (WCL) were immunoblotted using Flag-, HA-, and actin-specific antibodies, respectively. (B) Nemo−/− MEFs were transfected with HA-tagged IKK subunits and HA-tagged M45 or an unrelated MCMV control protein ( m142), respectively. IP and IB were performed using anti-HA and anti-Flag antibodies, respectively. WCL were immunoblotted with anti-NEMO antibody and as described for panel A. (C) rip1−/− MEFs were transfected with HA-tagged M45 or an unrelated MCMV control protein (m142) and Flag-tagged NEMO. IP was done with an anti-HA antibody. Immunoprecipitates and WCL were analyzed by immunoblotting using the indicated antibodies. (D) 293A cells were transfected with plasmids expressing HA-tagged M45 or m143 (as a control). Lysates were subjected to anti-HA immunoprecipitation. Immunoblotting of precipitates and the WCL was done with antibodies as indicated. (E) NIH-3T3 cells were infected for 4 hours with RM45 (MCMV expressing HA-tagged M45), ΔM45, or Rm142 (MCMV expressing HA-tagged m142) at an MOI of 5. Cell lysates were subjected to IP and IB with antibodies as indicated.
Figure 7A. Morphology of EBs grew for 8 days derived from individual or mixed ES cell lines. Hematoxylin and eosin staining. Scale bar, 100 μm. B. Quantification of Nanog expression in trisomic, wild-type, and mixed ES cell-derived EBs cultured for 8 days. Error bars, ±S.D. n=3. ** P < 0.01. C. The morphology of EBs at day 10 of differentiation. M15 was the normal EB formation condition and was used as control. Black arrows indicate the cystic structures. D. Boxplots of EBs diameter at day 10 of EB formation under M15, CM, and M15 supplemented with Bmp4 conditions. Each boxplot was plotted for more than 10 EBs. EBs cultured under CM or Bmp4 condition were compared to EBs of the same cell line cultured under M15 condition. * P < 0.05, ** P < 0.01.
Figure 1In the amyloidogenic pathway, the ectodomain shedding of the APP by BACE yields the immediate GSEC substrate, APPC99. GSEC endopeptidase cleavage of APPC99 generates APP intracellular domain (AICD50-99 or AICD49-99) and a 'de novo generated' Aβ substrate (Aβ48 or Aβ49, respectively). While the AICDs are released, the Aβ48 and Aβ49 are subjected to subsequent GSEC proteolysis (carboxypeptidase-like γ-cleavages). GSEC structure with co-purifying helical peptide (PDB: 5FN3) is presented. PSEN1 is shown in light brown, PEN-2 in dark brown, APH1A in gold, NCT in green and the co-purifying helical peptide in red. The topology of the helical peptide (putative GSEC substrate) is indicated by the amino and carboxyl terminal groups. The interface between the N-terminal part of the helical peptide, the NCT ectodomain, and PSEN1 is magnified and NCT/PSEN1 side chains shown.
Figure 4(A) A375 cells were infected with retroviruses expressing ZEB1. Western blot analyses of ZEB1 and p75. GAPDH was used as a loading control. (B) Quantitative PCR analyses of MITF, p75, ABCB5 and JARID1B upon ZEB1 expression. mRNA expression levels are represented relatively to control cells, in which the levels were fixed at 1 (mean ± SD, n=3, Student's t-test). (C) Soft agar colony formation assay upon ZEB1 expression. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test). (D) 2 x 106 Control or ZEB1-overexpressing A375 cells were injected subcutaneously into nude mice. The mean tumor volume for 5 mice is represented ± SEM (Student's t-test). (E) Western blot analyses of ZEB1, MITF, P-ERK, and TWIST1 levels in control or ZEB1-expressing cells +/- 150 nM PLX4032 treatment for 24 h. GAPDH was used as a loading control. (F) FACS analyses of p75 cell surface expression upon ZEB1 overexpression, after 10 days treatment with or without 150 nM PLX4032. Bar chart representing the mean percentage of p75-high, int and low cells from 2 independent experiments (Fisher's Exact Test). (G) Control or ZEB1-overexpressing A375 cells were transfected with control or p75-siRNA. p75 and MITF expression levels were was analyzed by quantitative PCR after 48 h. mRNA expression levels are represented relatively to control cells transfected with siRNA Control, in which the levels were fixed at 1 (mean ± SD, n=2). (H) Clonogenic assay +/- PLX4032 (150 nM), +/- GDC-0973 (5 nM) treatment for 10 days. The graphs represent the mean number of colonies (± SD) in 3 independent experiments (Student's t-test). (I) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or ZEB1-expressing cells (n=3, Student's t-test).
Actin remodelling is required for autophagosome–lysosome fusion under basal conditions.Figure 4(A) Wild‐type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies to Lamp‐1 (a lysosome marker, red) and ubiquitin (green) as indicated. F‐actin was detected by phalloidin (blue). The arrows indicate ubiquitin‐positive aggregates that are surrounded by F‐actin and Lamp‐1. (B, C) Wild‐type and HDAC6 KO MEFs were transfected with mCherry‐GFP‐LC3, followed by treatment with LatA (100 nM) or nocodazole (250 nM) for 6 h and analysed as described in Figure 3A. (D) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed mouse hepatocytes were treated or not with latrunculin (LatA) as indicated, extensively washed to remove traces of the inhibitor, and then labelled with the antibody and subjected to in vitro fusion assay in the presence or absence of purified actin. The number of total fusion events/total number of vesicles for each condition was as follows: 176/880; 233/1110; 84/930; and 180/950. The differences with untreated samples were significant at **P0.01. (E) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed or starved mouse hepatocytes were treated or not with latrunculin (LatA) as labelled and subjected to in vitro fusion assay. The differences with untreated samples were significant at *P0.05. The number of total fusion events/total number of vesicles for each condition was as follows: 169/1250; 87/972; 190/1120; and 175/1165. (F) Autophagosomes (APGs) and lysosomes (Lys) isolated from HDAC6 KO MEFs were treated or not with latrunculin (LatA) as indicated and subjected to in vitro fusion assay.
UPF3A overexpression does not affect NMD A Schematic representation of the bridge between UPF1 and the EJC during NMD.Figure 1A Schematic representation of the bridge between UPF1 and the EJC during NMD. Binding of UPF3A instead of the stronger bound UPF3B is discussed to either activate or repress NMD. B Western blot analyses after induced expression of FLAG-tagged UPF3A in WT HEK293 and HeLa cells (n=1). Tubulin serves as control. The asterisk indicates unspecific bands. C Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in HEK293 and HeLa WT cells overexpressing UPF3A using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR (ISAR) using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method. D Volcano plot showing the differential gene expression analyses from the RNA-Seq dataset of HEK293 and HeLa WT cells overexpressing UPF3A. The log2 fold change is plotted against the -log10 adjusted p-value (adj. p-value). P-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. OE = overexpression. E Read coverage of SMG5 from WT HEK293 and HeLa RNA-seq data with or without induced UPF3A overexpression shown as Integrative Genomics Viewer (IGV) snapshot. Differential gene expression (from DESeq2) is indicated as log2 fold change (log2FC) on the right. Schematic representation of the protein coding transcript below.
DFO leads to lysosomal degradation of ferritin.Figure 4(A) Cells were then incubated at the restrictive temperature (39°C) for 6 h in the presence or absence of 100 μM DFO, with or without 100 μM chloroquine or 10 μM MG132. Cells were then harvested and ferritin content determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate. (B) Cells treated as in (A) were incubated in the presence or absence of 5 mM 3‐methyladenine.
Figure 5A. Ubiquitination of FKBP38 depends on Parkin E3 activity. HeLa cells were transfected with the indicated vectors and treated with DMSO or CCCP (10 µM) for indicated times. MG132 (30 μM) was added 1 h before each treatment. Lysates of cells were subjected to an IP-IB assay with the indicated antibodies. WT, wild-type. B. Parkin fails to ubiquitinate the FKBP38 K271/273R mutant. HeLa cells were transfected with the indicated vectors and treated with CCCP (10 µM) for 30 h. MG132 (30 μM) was added 5 h after CCCP treatment. Lysates of cells were subjected to an IP-IB assay with the indicated antibodies and compared ubiquitination levels of WT, K271R, K273R, and K271/273R FLAG-FKBP38. C. Overexpression of WT FKBP38 is degraded in MITOL KO cells in mitophagy. MITOL KO HeLa cells stably expressing HA-Parkin were transfected with WT or K271/273R mutant FLAG-FKBP38 and treated with DMSO or CCCP (10 µM) for 24 h. Lysates of cells were subjected to an IB assay with the indicated antibodies. D. MITOL KO increases degradation of ER targeting FKBP38. HeLa cells stably expressing HA-Parkin were transfected with mitochondrial targeting FLAG-FKBP38 (FKBP38ActA) and ER targeting FLAG-FKBP38 (FKBP38IYFFT), and treated with DMSO or CCCP (10 µM) for 24 h. Lysates of cells were subjected to an IB assay with FLAG antibodies for organelle targeting FKBP38 or indicated antibodies. Another cell lysates were subsequently separated on a Phos-tag gel, followed by IB with anti-Parkin antibodies (Phos-tag). E-G. MITOL partly prevents CCCP-induced cell death by reducing Parkin-mediated degradation of FKBP38. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were transfected with the indicated vectors, and treated with DMSO or CCCP (10 µM) for 24 h. Cells were stained with propidium iodide (PI) for counting the percentage of dead cells using fluorescence microscopy. Data represent the mean ± SD of five independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. KR, FKBP38 K271/273R.
a) Cartoon of the FPP assay illustrating the cellular localization of ER-bound CD3δ-CFP (green) and YFP-CD3δ (red), and the position of the fluorescent protein tag relative to its respective membrane.figf3(b) NRK cells expressing CD3δ-CFP and YFP-CD3δ were subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 4 mM trypsin as indicated. Scale bar, 10 μm. c) Biochemical protease protection assay of NRK cells expressing YFP-CD3δ, CD3δ-CFP and GFP. Transfected cells were subjected to SDS-PAGE and Western blot analysis before (lane 1) and after separation of the cytosolic (lane 2) and membrane (lane 3) fractions by ultracentrifugation. Equal amounts of the membrane fraction were treated with trypsin in the absence (lane 4) or presence (lane 5) of Triton X-100. Protein extracts were analyzed by immunoblotting with monoclonal anti-GFP antibody JL-8.
The loop structure in the CbpC dp3 domain enabled interaction with the CCD of Atg14.Figure 4A Diagram of the Atg14 derivatives used in (B). B GST-pulldown assays performed using GST-CbpFR6 or GST and lysates from 293T cells expressing GFP-Atg14 derivatives or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody. C Schematic representation of the CbpFR6 derivatives used in (D) and (E). The domains are color-coded according to the crystal structure report for CbpFR6 (lower left) [10]. D, E GST-pulldown assays using GST-CbpFR6 derivatives and lysates from 293T cells expressing GFP-Atg14 CCD or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody. Each GST-CbpFR6 derivative-bound bead was confirmed by Coomassie brilliant blue (CBB) staining. F Structure of CbpFR6 (PDB ID: 2V04). The overall structure (left) and dp3 domain (right) of homology-modeled structures are shown. The side chains of Y83 and E95 and bound choline molecules are shown as green and yellow sticks, respectively. G The amino acid sequences of the CbpCT4/CbpFR6 mutants used in (H), (I), and (J). H GST-pulldown assays performed using GST-CbpFR6 mutants or GST and lysates from 293T cells expressing GFP-Atg14 CCD or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody. Each GST-CbpFR6 mutant-bound bead was confirmed by CBB staining. I Lysates of 293T cells transiently expressing GFP-CbpCT4 mutants or GFP were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin. J Confocal microscopy images of HeLa cells transiently expressing the indicated GFP-CbpCT4 mutants. Scale bars, 10 µm.
Figure 5A. Schematic depicting competition assay using GFP-LukE and unlabeled WT or mutant toxins. B. Competition assay using GFP-LukE (312.5 nM) and a dose response of unlabeled WT or dominant-negative (DN) toxin subunits evaluated by flow cytometry on the surface of primary human PMNs. E = LukE, Emut1 = LukEmut1, A = HlgA, Amut1 = HlgAmut1, S = LukS-PV, C = HlgC. Data is presented as total fluorescence emitted from PMNs. A representative of three independent experiments each done with hPMNs isolated from different donors is shown. C. Schematic depicting recruitment assay using GFP-LukD and unlabeled WT or dominant-negative toxins. D. Recruitment assay using GFP-LukD (156.3 nM) and a dose response of unlabeled dominant-negative (DN) toxin subunits evaluated by flow cytometry on the surface of primary human PMNs. Emut1 = LukEmut1, Amut1 = HlgAmut1. Data is presented as total fluorescence emitted from GFP-positive PMNs. A representative of three independent experiments each done with hPMNs isolated from different donors is shown. E. Immunoblot using anti-His antisera to detect dimer and oligomer formation by purified polyhistidine (6xHis)-tagged WT or dominant-negative LukED toxins in the absence or presence of 2-methyl-2,4-pentanediol (MPD, 40% v/v). Emut1 = LukEmut1, Dmut = LukDmut. A representative of two independent experiments is shown.
Figure 2Dot plots summarizing the transcriptomics levels of ACE2 based on different scRNA-seq datasets and tissues. Three different scales were used, in order to be able to compare percentages of cells expressing ACE2 both between and within tissue types. The size of the dots indicates the percentage of cells expressing ACE2 in respective cell type with a maximum of 60% (left column), 10% (middle column) and 1% (right column), and the color saturation corresponds to the average expression level. Plots were generated using Seurat package in R.
Box-Whisker plots of lysosomal acidity quantified by AO fluorescence microscopy and image analysis.Figure 3  Acridine Orange was added to living untreated ASPC-1 and MiaPaCa-2 cells and cells treated with 5-FU , OMP or the combination of both for 30 minutes or 24 hours. Microscopical life images were taken at 525 nm (green) and 650 nm (red) to detect changes in the lysosomal pH value (three images per plate, three plates per group). The red to green fluorescence ratio of the lysosomes of treated cells were compared to the control groups by the Mann-Whitney-U-test. Significant differences compared to control are marked by *. In ASPC-1 cells, after 30 min of treatment, intralysosomal acidity increased upon treatment with OMP (p:0.0051) and 5-FU+OMP (p<0.0001). After 24 hours, the acidity is increased upon all treatment regimens (5-FU - p:0.0002; OMP - p<0.00001; OMP+5-FU - p:0.037). In MiaPaCa-2 cells the acidity is elevated after 30 min upon 5-FU (p:0.005) and decreased after treatment with OMP (p:0.037), 5-FU (p:0.00026) and 5-FU+OMP (p:0.011) after 24 hours.
Figure 1a) Auto-ubiquitination assays in which the E3 acts as both an E3 and as proxy substrate were performed with GST-HHARIRBR, T7-Triad1ΔAri, GST-ParkinRBR, and Flag-BRCA1/BARD1 and either UbcH5WT or UbcH5L104Q as the E2. Products were visualized by western blotting against indicated tags on E3s. Times given are post-ATP addition. b) Overlay of (1H,15N)-HSQC-TROSY spectra of 15N-UbcH5-O-15N-Ub in the absence (black) and presence (red) of 0.5mol equiv. HHARI RING1. A subset of UbcH5 peaks, but not Ub peaks, shift and broaden upon HHARI RING1 binding. c) CSPs from (b) are mapped onto the structures of UbcH5c (PDB 2fuh) and Ub (PDB 1ubq). Residues that exhibit loss of intensity > 1 stdv upon RING1 binding (intensity < 0. 47) are colored on each structure: UbcH5 residues 5, 6, 7, 16, 20, 22, 56, 62, 74, 87, 90, 91, 96, 97, 99-103, 137, 138 (pink) and Ub residues 48, 50 (yellow). The lack of CSPs on the UbcH5 cross-over helix and on the surface of Ub signifies that HHARI RING1 does not induce closed UbcH5~Ub. d) CLUSTAL OMEGA alignments of the C-terminal sequences of RBR RING1 domains (top) and canonical RING domains (bottom). Each alignment includes the 7th and 8th Zn2+ coordinating positions (gray). The allosteric linchpin position critical for E2~Ub activation by RING-type E3s is highlighted in yellow. RBR RING1 domains lack the allosteric linchpin.
TFEB interacts with active Rag heterodimers.Figure 2.(A) ARPE-19 cells were nucleofected with the indicated Rag-expressing plasmids. 6 h after nucleofection, cells were infected with adenovirus expressing TFEB-FLAG-WT. 12 h later, cells were lysed and subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against Rag proteins and FLAG (used to detect TFEB-WT). (B) ARPE-19 cells were nucleofected with the indicated Rag- and TFEB-GFP-expressing plasmids. 18 h later, cells were lysed and subjected to immunoprecipitation with the anti-HA antibody (used to immunoprecipitate Rag proteins). The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and GFP (used to detect Rag proteins and TFEB-WT, respectively). (C) ARPE-19 coexpressing TFEB and the indicated Rag-expressing plasmids were lysed and subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and FLAG (used to detect Rag proteins and TFEB-WT, respectively). (D) Immunoblotting analysis of coimmunoprecipitated TFEB-FLAG-S211A with Rag heterodimers. IP, immunoprecipitation.
Figure 1CRTH2 subcellular localization in GFP-fused CRTH2 expressing plasmid-transfected cells. Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane. Green, GFP-CRTH2; blue, DAPI; red, Calnexin; Scale bar, 20 μm. Western blot analysis of CRTH2 expression in cell membrane and ER in NIH 3T3 and HEK293T cells. Myc-tagged CRTH2 expressing plasmid was transfected into NIH 3T3 and HEK293T cells. Whole cell lysate, plasma, and ER fractions were obtained 48 h after transfection. Calnexin and ATP1B were used as markers for ER and cell membrane, respectively. The effect of TGF-β1 (10 ng/mL) on the expression of caveolin-1 in NIH 3T3 cells. Representative images of CRTH2 subcellular localization in NIH 3T3 cells in response to TGF-β1 (10 ng/mL) treatment. Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane. Green, GFP-CRTH2; blue, DAPI; red, Calnexin; Scale bar, 20 μm. Quantification of CRTH2 colocalization with endoplasmic reticulum after TGF-β1 or vehicle treatment. *P < 0.05 vs vehicle (Mann-Whitney U test); n = 4 for all groups. Data are expressed as the mean ± standard error of the mean.
Sphinganine exposure activates molecular signatures of genome instability and senescence in human cardiomyocytes while concomitant HAT inhibition amends the genotoxicity: Gene Ontology (GO) enrichment status for the differentially expressed transcripts upon DHS treatment.Figure 5Gene Ontology (GO) enrichment status for the differentially expressed transcripts upon DHS treatment. The bar graphs represent GO enrichment levels for (A) biological processes (B) clinical phenotypes. (C) Co-treatment of curcumin lead to the activation of DNA repair pathways as shown here by functional enrichment analysis on the differentially expressed transcripts using Metascape. (D) Heatmap depicting the expression profile of the markers of DNA damage response in the indicated conditions. (E) Co-treatment of curcumin activates DNA repair pathways as depicted here by heatmaps.
a,b) HeLa cells were treated with 10 μg ml−1 N-Shh peptide for 24 h.f1(a,b) HeLa cells were treated with 10 μg ml−1 N-Shh peptide for 24 h. (a) Endogenous LC3 was detected by immunoblot in total cell lysates, quantified by densitometric analysis and normalized to actin. Relative levels were expressed in percentage with control cells set to 100 and shown in graph. (a,b) HeLa cells were treated with 10 μg ml−1 N-Shh peptide for 24 h.(b) mRNA was obtained and levels of Ptch1 or Gli1 and actin mRNA were amplified by standard PCR and visualized in agarose gels. Graph shows mRNA levels quantified by densitometric analysis and normalized to actin. (c) 8xGli-luciferase construct was transfected in HeLa cells for 24 h, followed by treatment with 10 μM purmorphamine, or DMSO as a control, for another 24 h. Graph represents the mean value of the firefly luciferase activity relative to Renilla transfection control. (d) Endogenous LC3-II was detected in cell lysates from cells treated with 10 μM purmorphamine for 24 h, either in the absence or presence of trehalose (100 mM). Where indicated, bafA1 (400 nM) was added for the last 4 h. Quantification by densitometric analysis relative to actin is shown in the graph. (e) Cells were treated with 10 μM purmorphamine for 24 h under starvation conditions, either in the presence of bafA1 or DMSO as a control, and LC3-II and actin levels were detected. (f) HA-HttQ74 construct was transfected into HeLa cells followed by treatment with 10 μM purmorphamine for 24 h. The percentage of transfected cells with aggregates detected by HA immunofluorescence is shown in the graph. P-values were calculated by odds ratio. (g) U20S cells stably expressing HaloTag-p62 were labelled with HaloTag ligand for 15 min and washed out followed by treatment with purmorphamine. After 48 h, cells were lysed, run on a SDS-polyacrylamide gel electrophoresis and fluorescent HaloTag was visualized using a Typhoon 8600 variable mode imager. Fluorescence was normalized to total protein levels, detected on the same gels by Kryton Fluorescence protein stain. A representative gel and its quantitated normalized levels are shown. In all panels, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student's t-test unless indicated: ***P0.001; **P0.01; *P0.05. SDS-PAGE, SDS-polyacrylamide gel electrophoresis.