EMBO/sd-character-level-ner huggingface dataset:
Figure 2Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala wMelOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala BurkOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. Note that the rise in fluorescence polarization signal, indicative of a noncovalent interaction, is specific to the Ub substrate. Heatmap representation of corrected OTU activities toward the Ub and Ub-like fluorescent substrates. In the reactions marked by an asterisk, an unusually high level of noise in fluorescence polarization signal was observed, likely a result of high OTU concentration. Ub chain specificity assay measuring wMelOTU activity toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS-PAGE, and visualized by Coomassie staining. Ub chain specificity assay measuring BurkOTU activity toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS-PAGE, and visualized by Coomassie staining. Heatmap representation of WT bacterial OTU activities toward the eight diUb linkages at the indicated timepoints.
Fig.Figure 3C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and expression of Sox14 was assessed by immunofluorescence. A - C Sox14 stainings at 2 h APF. (A) Control c4da neurons. (B) C4da neurons expressing PP2A-29B dsRNA. (C) C4da neuron expressing dominant-negative Mts. D Quantification of Sox14 expression in (A - C). C4da neuron Sox14 expression was normalized to Sox14 expression in neighboring ddaE c1da neurons (identified by characteristic cell body shape). Data are mean +/- s. d.. The numbers of neurons (animals) for each genotype are given in each graph (data were not derived from independent experiments). n. s., not significant, *** P<0.0005, Wilcoxon's test. C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and expression of Mical was assessed by immunofluorescence. E - H Mical stainings at the indicated time points. (E) Mical expression in control c4da neurons at 2 h APF. (F) Mical expression in c4da neurons expressing dominant-negative Mts at 2 h APF. (G) Mical expression in c4da neurons expressing PP2A-29B dsRNA at 2 h APF. (H) Mical expression in c4da neurons expressing Orco dsRNA at 0 h APF. (I) Mical expression in c4da neurons expressing PP2A-29B dsRNA at 0 h APF. J Quantification of Mical expression in (E - I). C4da neuron Mical expression was normalized to Mical expression in neighboring ddaE c1da neurons (marked by asterisks). Data are mean +/- s. d.. The numbers of neurons (animals) for each genotype are given in each graph (data were not derived from independent experiments). n. s., not significant, ** P<0.005, *** P<0.0005, Wilcoxon's test. Data information: Scale bars in A and E are 10 μm.
Fig.Figure 7(A) Examples of the body sizes of the WT, L-HRD KO and HRD1/FGF21 DKO mice. (B, C) Body weight and body height of the WT, L-HRD KO and HRD1/FGF21 DKO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test. (D) Tibia lengths of the WT, L-HRD KO and HRD1/FGF21 DKO mice fed with normal chow diet. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test. (E) Hepatic Hsd3b5, Cyp2d9 and Cyp4a12 mRNA in the WT, L-HRD1 KO and HRD1/FGF21 DKO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test. (F) Estrus cycle of L-HRD KO and HRD1/FGF21 DKO mice at the age of 4 months. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test. (G) Percentage of the Activity, feeding and water intake of the WT, L-HRD KO and HRD1/FGF21 DKO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.
FoxO3a poises HSCs for rapid autophagy induction.Figure 3Status of the autophagy machinery in WT HSCs (n = 3). Results are expressed as log2 fold expression compared to WT GMPs (set to 0). b, c, qRT-PCR analyses of pro-autophagic genes in WT (b) and FoxO3a−/− (c) HSCs (n = 3). Results are expressed as log2 fold expression compared to WT GMPs (b) or FoxO3a+/+ HSCs (c) (set to 0). Autophagy flux in cultured FoxO3a−/−::Gfp-Lc3 HSCs (n = 3). Results are expressed as percent GFP-LC3 MFI in -BafA vs. +BafA conditions, and normalized to FoxO3a+/+−::Gfp-Lc3 HSCs. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Figure 4ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant µs chains and Halo-ERp44, or Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls. A. Aliquots from the spent media (Extracellular) were blotted under non-reducing conditions and decorated with anti-µ antibodies. The blue arrow points at the faster migrating µs2L2 released by cells expressing wild type µs or L566A chains . Expression of HaloERp44 restores C575-dependent retention, and wild type µs are secreted only as polymers (black arrow). Traces of covalent HaloERp44-µ2L2 complexes (black asterisks) are detected in the media of cells expressing HaloERp44∆RDEL and µs or L566A. The slightly faster mobility of µs2L2 and µsL complexes released by cells expressing ERp44∆RDEL suggest that the latter inhibits processing of the N563 glycan, likely hindering it to the Golgi enzymes upon non-covalent binding. Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition. ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant µs chains and Halo-ERp44, or Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls. B. The corresponding cell lysates are shown in this panel. The short black arrow points at the polymers formed in ERp44KO HeLa cells upon expression of wild type HaloERp44. The black asterisks indicate covalent complexes that this transgene forms with C575 containing µ2L2. Traces of intracellular µs2L2-ERp44∆RDEL complexes are visible in µs and L566A but absent from C575A expressing cells. Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition. C. Lysates of the ERp44KO transfectants were precipitated with NP-sepharose, resolved under non reducing conditions and the blot decorated sequentially with anti-µ, anti-ERp44 or anti-Halo. The two black asterisks point at bands corresponding to covalent complexes containing µs2L2 and endogenous or Halo-tagged ERp44.
Figure 7Cell viability measured by CellTiter-Glo in Calu6 parental and Calu6-VAR cells treated with different concentrations of volasertib for 72 hours. Micrograph shows acquisition of a mesenchymal-to-epithelial transition phenotype in VAR cells. Basal protein and mRNA expression in parental and VAR cells for the indicated proteins was measured using immunoblotting (upper) and reverse-transcriptase PCR (lower), with GAPDH expression used for normalization. Parental and VAR cells were treated as indicated for 48 hours and apoptosis was measured using the Apo-BrdU assay. Parental and VAR cells were treated with the indicated drug concentrations for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins. Parental and VAR cells were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ.
Identification of Nanog as the stemness factor in KL lung tumors.Figure 1(A, Gene Set Enrichment Analysis (GSEA) identifies Stem-cell like expression signature in KL tumors compared with KrasG12D/+ (K) lung tumor (A) Significance was calculated by permutation test. B) Gene Set Enrichment Analysis (GSEA) identifies Stem-cell like expression signature in KL tumors compared with KrasG12D/+; P53-/- (KP) lung tumors (B). Significance was calculated by permutation test. (C) Heat map demonstration of genes that were mostly enriched in the pathway of pluripotency of stem cells. The color bar showed the relative RNA-seq signal (Z-score of normalized FPKM). (D) The protein abundance of NANOG was upregulated in KL tumors compared with K and KP mice. Scale bar, 50 μm. (E) Scheme illustrator of mouse model. Histological examinations of tumor types in (F) K versus KrasG12D; NanogL/L (KN) Tumor tissues collected form indicated mice analyzed using Haematoxylin Eosin (H&E) staining. Representative H&E images are shown. Scale bar, 500 μm (top), 50 μm (bottom). Histological examinations of tumor types in (G) KP versus KrasG12D; P53L/L; NanogL/L (KPN) Tumor tissues collected form indicated mice analyzed using Haematoxylin Eosin (H&E) staining. Representative H&E images are shown. Scale bar, 500 μm (top), 50 μm (bottom). Histological examinations of tumor types in (H) KL versus KrasG12D; Lkb1L/L; NanogL/L (KLN). Tumor tissues collected form indicated mice analyzed using Haematoxylin Eosin (H&E) staining. Representative H&E images are shown. Scale bar, 500 μm (top), 50 μm (bottom).
Prom1 disruption ameliorates glucagon-induced hyperglycemia in DIO mice.Figure 7(A) Wild type primary hepatocytes were infected with an adenovirus harboring sh-control (sh-Con), sh-radixin (sh-Rdx), GFP (Ad-GFP), and/or sh-resistant wildtype radixin (Ad-RdxR) or AKAP-dead LPTD mutant of radixin (Ad-LPTDR) for 24 hours. (B) Increasing amount of Ad-RdxR or Ad-LPTDR were infected to WT primary hepatocytes for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. Levels of p-PKA substrates, p-CREB, CREB, radixin, Myc-radixin and GAPDH were determined by immunoblotting. Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (C) DIO WT (Prom1+/+) and KO (Prom1-/-) mice were intraperitoneally injected with glucagon. Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon (100 µg/kg body weight) stimulation (n = 10 mice/ group). Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (D) Levels of p-PKA substrates, p-CREB, CREB, Prom1 and GAPDH were determined by immunoblotting 10 minutes after glucagon (2 mg/kg body weight) stimulation (n = 3 mice/group). Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (E) Glucose disposal rates in DIO WT (Prom1+/+) and KO (Prom1-/-) mice were measured using glucose and insulin tolerance tests (n = 10 mice/group). Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (F) DIO KO (Prom1-/-) mice were infected with an adenovirus harboring LacZ or Prom1 for 24 hours, fasted for 4 hours and intraperitoneally injected with glucagon (100 µg/kg body weight). Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (GST). Glucose disposal rates were measured using the pyruvate tolerance test (PTT) (n = 10 mice/group).
Figure 2Schematic representation of a full-length experiment Schematic representation of Aβ42 oligomers (right panel) and APOE4 (left panel) binding with soluble TREM2-Fc at various concentrations. Curves were fit by setting buffer control (no ligand) to y=0 Calculated Kon, Kdis and KD values for Aβ42 oligomers or Aβ42 monomers, Aβ40 monomers, APOE3 or APOE4 association/dissociation with soluble TREM2-Fc and AD variants. Kinetic constants were calculated using five different concentrations of ligand and the entire experiment (including new ligand preparations and purifications of soluble TREM-Fc) was repeated twice (# p < 0.05, ANOVA, Tukey's Multiple Comparison Test, see Appendix table S1 for p value) Calculated kon, Kdis and KD value for Aβ42 oligomers association/dissociation with soluble human vs. mouse TREM-Fc family members. Kinetic constants were calculated using five different concentrations of ligand and the entire experiment (including new ligand preparations and purifications of soluble TREM-Fc) was repeated twic
Fig.Figure 2A Schematic for timing of transgene expression and start of live imaging. Gene expression was induced at the end of the second larval instar, laser ablation and imaging started 30h later. B Wound healing in epithelia with clonally expressed cytoplasmic GFP (magenta) under the actin5c-Gal4 driver in control larvae (act>GFP) or larvae expressing RNAi constructs specific for Atg1 or Atg5. Laser ablated cells are marked with white asterisks. To visualize cell borders DE-cad-RFP (green) was expressed in all tested genotypes. By the end of wound closure, GFP from the clonal cells has spread to all cells around the wound in the control, but not if autophagy is suppressed, regardless of the number of cells initially expressing GFP. C Control larva in which a GFP-expressing cell was wounded. No GFP is induced in or taken up by the surrounding cells. D Control experiment in which the central cell was damaged but not killed (white marked area). No wound response occurs and no GFP-leakage between neighbouring cells is seen.
Figure 1A) gfp::znfx-1; pgl-1::tagrfp animals were mutagenized with ethyl methanesulfonate (EMS). F2 progeny of mutagenized animals were fed bacteria expressing dpy-11 dsRNA. Mutant animals that failed to inherit dpy-11 RNAi (were non-Dumpy) were isolated. Lineages established from these animals were examined under a 63X oil objective on a Zeiss Axioplan fluorescence microscope for defects in number, size, morphology or subcellular distribution of Z granules using GFP::ZNFX-1 as a marker of Z granules. B) Fluorescence micrographs of germlines from animals expressing GFP::ZNFX-1 and PGL-1::TagRFP. The three panels on the left show about a dozen pachytene stage nuclei from a control (starting strain) animal, or the three classes of mutation identified in the genetic screen. Scale bar, 5μm. For class III images, nuclei are outlined with a dotted white line for clarity. The far right panel (GFP::ZNFX-1 inset) shows a blow-up of one nucleus/granule from each genotype to better illustrate the Z granule defects. Scale bar for insets: 2μm.
Rpd3 and other members of the Rpd3L complex negatively regulate H3K79 methylation.Figure 1Schematic overview of the Epi-ID strategy. Epi-ID H3K79 methylation scores of the deletion mutants of members of the Rpd3L and Rpd3S complexes, calculated as described in Appendix Supplementary Methods, where 0 means a wild-type H3K79me level (log2 scale). The gray dots indicate accessory subunits. UpTag and DownTag are barcode reporters in a promoter and terminator context, respectively. Data was obtained on all Rpd3L/Rpd3S subunits except Sds3. Mass spectrometry analysis of H3K79 methylation in wild-type and mutant strains. Mean and individual data points of two biological replicates. p<0.05 (*), p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type. Heatmaps of H3K79me1, H3K79me3, H2Bub and H3 in wild-type cells, aligned on the TSS. Genes were sorted based on the average H3K79me3/H3K79me1 ratio in the first 500 bp. Snapshot of depth-normalized ChIP-seq data tracks from wild-type and rpd3Δ strains showing 6 kb surrounding the DBP1 ORF, which is the top gene in the heatmap in panel (F). All tracks have the same y axis (0-20 RPM). A snapshot of another top-regulated gene is shown in Fig EV1D. Heatmap of the H3K79me3/H3 change in rpd3Δ versus wild-type cells, aligned on the TSS. Genes were sorted based on the average ratio in the first 500 bp.
Figure 4A. Differentiated A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h. B. Differentiated NT, c-src KD #1 or c-src KD#2/A53T α-syn-EGFP OE SH-SY5Y cells were cultured 12 h. Levels of α-syn in culture media were then measured using ELISA. Values were derived from three independent experiments (n=3). *** P < 0.001, ** P < 0.01 compared to control, one-way ANOVA. C. Schematic diagram showing the dual chamber assay. D. SH-SY5Y cells in the lower chamber were cocultured with differentiated NT, c-src KD #1 or c-src KD #2/ A53T α-syn-EGFP OE SH-SY5Y cells for 12 h. The cells in the lower chamber were immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P < 0.001 compared to NT cells, one-way ANOVA. E. Differentiated A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 5 μM saracatinib (sara) or 10 μM SKI-1 in the presence of DMSO or 50 μM bafilomycin A1 (BafA) for 12 h. Differentiated NT and c-src KD/A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 50 μM BafA for 12 h. Western blot was performed. Data are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA. G. NT and c-src KD primary neurons were incubated with 50 μM BafA for 12 h. Western blot was performed. Data are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA. H. SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of DMSO or 50 μM BafA for 12 h. The cells were immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). ** P < 0.01, one-way ANOVA.
Figure 4(A) Sequence alignment of COP1 pseudosubstrate latch with the COP1 4A mutant and COP1-binding motifs from previously identified COP1 binding partners. Residues in the sequences are colored to indicate residues conserved in the COP1-binding motif: Val-Pro dipeptide (red), acidic residues (blue), conserved Gln (magenta). (B) Representative images of COS7 cells transiently transfected with GFP-COP1 WT or GFP-COP1 4A (green). Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. (C) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 in (B). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 WT was calculated using the Student t test (****p < 0.00001). (D) Fluorescence polarization competition assay measuring the ability of unlabeled COP1 PSL, COP1 PSL 4A, and TRIB1 tail peptides to compete with FITC-TRIB1 tail for binding to the COP1 WD40 domain (376-731). The COP1 WD40 domain was preincubated with FITC-TRIB1 tail before addition of the indicated concentrations of unlabeled peptide. The change in polarization is plotted as a function of unlabeled peptide concentration. Curves were fit to a sigmoidal dose-response curve. Data are presented as mean values ± S.E.M. for three independent experiments where each measurement was made in duplicate.. (E) Surface representation of the crystal structure (PDB: 5IGQ) of the COP1 WD40 domain colored according to electrostatic surface potential, calculated by APBS (Baker et al, 2001), in complex with the TRIB1 tail (cyan). Inset shows zoomed view of TRIB1 Q356 inserting its sidechain into the central channel of the COP1 WD40 domain. (F) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green) and empty vector or TRIB1-FLAG (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. (G) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (F). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance was calculated using the Student t test (ns, not significant, ***p < 0.0001; ****p < 0.00001).
Composition of oskar mRNPs ex vivo.Figure 2(A-B") Colocalization of oskMS2-mCherry (red, A, B) (A', B') with Khc-EGFP (green, A) (A') or with EmGFP-Tm1-I (green B) (B') in ex vivo ooplasmic preparations.(C) Fraction of oskMS2-MCP-mCherry mRNPs located non-randomly within a 200 nm distance of one of the indicated GFP tagged protein particles in ex vivo ooplasmic preparations. MCP indicates MCP-EGFP which, like MCP-mCherry, can bind to MS2 loops. Staufen (Stau) is a dsRNA binding protein and bona fide partner of oskar mRNA (St Johnston et al., 1991; St Johnston et al., 1992). All values are significantly different from zero (p<10-3, one sample t-test).(D) Fraction of oskMS2-mCherry mRNPs co-localizing (max. 200 nm) non-randomly with Khc-EGFP particles in wild-type and in Tm1eg1/Tm1eg9 ooplasms in presence of two (white) or one (grey) copy of endogenous Khc.(C, D) P values of two sample t-tests are indicated above the relevant bar pairs. Numbers indicate the number of particle clusters (160 mRNPs in each) and the number of preparations (in brackets) analysed. Error bars represent 95% confidence intervals.
Figure 6Calu6, H157, and H1355 TPR-Met-expressing or vector control (pBABE)-expressing cells were treated as indicated for 24 hours and apoptosis was measured using the Apo-BrdU assay. Parental and TPR-Met-expressing cell lines were treated with the indicated drugs for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins. β-actin was used as a loading control. Parental and TPR-Met-expressing cell lines were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ. Data are means ± standard error of the mean from three independent experiments. Significant differences using two-way analysis of variance with Bonferroni or Benjamini-Hochberg correction for multiple comparisons are indicated. Mesenchymal NSCLC cell lines with the noted MET alterations were incubated with 50nM volasertib for four hours and subjected to immunoblotting with the indicated antibodies. *p < 0.05. **p < 0.01.