EMBO/sd-character-level-ner huggingface dataset:
Ykt6 phosphorylation affects early and late steps in autophagy.Figure 3A) ykt6ts cells containing centromeric plasmids as indicated were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 ºC for 1 hour before starvation for 1 hour at 37 ºC. Cells were lysed and the total extract (T) separated into a cytoplasmic (S) and a 100'000x g membrane pellet (P) fraction. Fractions were analyzed by anti-HA, anti-Tom70 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown. B) vam3∆ or ykt6ts vam3∆ cells containing the indicated centromeric plasmids were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 °C for 1 hour followed by 1 hour starvation in SD-N at 37 ºC. After cell lysis, samples were subjected to proteinase K (ProtK) and Triton X-100 (TX100) treatment as indicated and analyzed by anti-Ape1 Western blotting. Ape1*: proteinase K-resistant fragment of Ape1. One representative experiment out of three biological replicates is shown. C) Trichloroacetic acid extracts were prepared from ykt6ts cells containing an empty plasmid, 3HA-Ykt6 wild type, or 3HA-tagged Ykt6 mutant variants. Cells were grown at the permissive temperature (23 ºC) to late exponential phase in selective medium, shifted to the restrictive temperature (37 °C) for 1 h preincubation and continued to grow at the restrictive temperature for another 4 hours before harvesting. Samples were analyzed by anti-CPY and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown. D) Exponentially growing ykt6ts cells at permissive temperature (23 ºC) containing centromeric plasmids as indicated were shifted to the restrictive temperature (37 ºC) for 1 hour before starvation for 4 hours in SD-N. Trichloroacetic acid extracts were prepared. Lipidation of Atg8 was analyzed on a 15 % SDS-PAGE containing 6 M urea by anti-Atg8 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.
AIN‐1 colocalizes with SQST‐1 aggregates in autophagy mutants and directly interacts with LGG‐1.Figure 4(A-D) Both AIN‐1::GFP and SQST‐1, detected by anti‐SQST‐1 antibody, are very weakly expressed and diffusely localized in wild‐type embryos. (E-H) AIN‐1::GFP aggregates colocalize with SQST‐1 aggregates in atg‐3 mutant embryos. Scale bars, 10 μm for insets that show a magnified view. (I-L) AIN‐1::GFP aggregates are separable from PGL granules, detected by anti‐SEPA‐1, in atg‐3 mutant embryos. (M-P) Colocalization of AIN‐1::GFP aggregates with LGG‐1 puncta in epg‐6 mutant embryos. (Q) % of AIN‐1::GFP aggregates colocalized with SQST‐1 aggregates, SEPA‐1 aggregates and LGG‐1 puncta in autophagy mutants. Error bars indicate the s.d. of four examined embryos. (R) A co‐IP assay shows that AIN‐1 associates with LGG‐1 in vivo. (S) Direct interaction between AIN‐1 and LGG‐1 in an in vitro pull‐down assay. 5% of the His fusion proteins used for pull‐down are shown as input. Corresponding Ponceau‐stained blots show the amount of proteins used for pull‐down. (T) A fragment of AIN‐1 directly binds to LGG‐1 in an in vitro pull‐down assay. (U) Model for the role of autophagy in modulating the miRNA pathway. co‐IP, coimmunoprecipitation; GFP, green fluorescent protein; MBP, maltose‐binding protein; miRNA, microRNA; IP, immunoprecipitant; RISC, RNA‐induced silencing complex; s.d., standard deviation.
pex3 atg1 cells harbor Pex14-containing structures.Figure 1.(A) EM analysis of KMnO4-fixed pex3 atg1 and WT cells grown for 16 h on MM-M/G. The inset shows a cluster of vesicles (enlarged from the boxed region). (B) iEM analysis of pex3 atg1 cells using α-Pex14 antibodies. (C and D) FM images of pex3 atg1 cells producing Pex14-mCherry and the ER marker BiPN30-eGFP-HDEL (C), or Pex14-mGFP complemented with Mitotracker orange staining (D). (E) Electron tomography analysis of a serial-sectioned pex3 atg1 cell (a) containing a perinuclear membrane cluster (b, arrows). (d-f) 10-nm-thin digital slices through the tomogram reconstruction (viewing direction indicated in c) revealed vesicles (black arrows) and reticular structures (red arrows). The surface-rendered reconstruction in g shows the reticulovesicular structures in 3D viewed at right angles from d. CW, cell wall; M, mitochondrion; N, nucleus; P, peroxisome; V, vacuole. Bars: (A) 500 nm; (A, inset; and B) 100 nm; (C and D) 2.5 µm; (E) 250 nm.
in human and murine cells (A) Human myeloid cells and lymphoid cells were purified from PBMCs using a cell sorter.Figure 1(A) Human myeloid cells and lymphoid cells were purified from PBMCs using a cell sorter. Total RNA was isolated from these primary cells and primary AECs to perform chip hybridization and microarray analysis. The profile of EphA2 expression in different cells is indicated. (B) Murine B cells and T cells were purified from spleens using a cell sorter, total RNA was isolated from murine B cells, T cells, BMDCs, BMDMs, AMs and AECs. Shown is mean ± SD. Data represent three independent biological replicates. The profile of EphA2 expression in different cells is indicated. (C) Immunoblot analysis of EphA2 and β-actin in BMDCs, BMDMs, AMs, and AECs.
a) Western blot analysis showing the replacement of endogenous Beclin 1 with Beclin 1–EGFP in Becn1−/−;Becn1–EGFP/+ mice, as detected by an anti-Beclin 1 antibody.figf1(a) Western blot analysis showing the replacement of endogenous Beclin 1 with Beclin 1-EGFP in Becn1-/-;Becn1-EGFP/+ mice, as detected by an anti-Beclin 1 antibody. (b) Coomassie-stained SDS-PAGE showing the Beclin 1-interacting proteins immuno-isolated from brain and liver of the 'rescued' mice (lanes 2 and 4) and of control Becn1+/- littermates (lanes 1 and 3), using an anti-GFP antibody. Proteins in the gel bands were extracted and identified by mass spectrometry as Vps15/p150 (band 1), Vps34/class III PI(3) K (band 3), UVRAG (band 4), Beclin 1-EGFP (band 5), Atg14L (band 6, asterisk, gi|27369860) and Rubicon (band 2, asterisk, gi|45708948). UVRAG levels varied with different affinity-purification conditions, suggesting an unstable association of UVRAG with the complex. (c) Schematic representations of the domain structures of Atg14L and Rubicon. Atg14L contains two coiled-coil domains (CCD1 and CCD2), which are also homologous with the SMC domain (structural maintenance of chromosomes). Rubicon contains an N-terminal RUN (for RPIP8, UNC-14 and NESCA) domain, a C-terminal Cys-rich domain and a central CCD domain. (d) Western blot analysis of Atg14L, Rubicon, Vps34 and Beclin 1 in gel filtration fractions from wild-type mouse liver extract showed co-elution of these proteins in fractions 38-45. Atg14L was also eluted in later fractions 51-56. The fractions for the peak elution of thyroglobulin (670K) and γ-globulin (158K) are indicated by arrows. Control siRNA-transfected NIH 3T3 cell lysate was loaded as a positive control (labelled as 1) for the migration position of the Atg14L protein on SDS-PAGE; Atg14L siRNA-transfected NIH 3T3 cell lysate was loaded as a negative control (labelled as 2). (e, f) Co-immunoprecipitation confirmed protein-protein interaction between Atg14L and Rubicon. HEK 293 cells were co-transfected with Atg14L-EGFP and Flag-Rubicon (e) or Rubicon-EGFP and Flag-Atg14L (f). Cell lysates were used for immunoprecipitation with an anti-GFP antibody and the resulting immunoprecipitates were blotted with an anti-Flag antibody. Our results show immunoprecipitation of Rubicon by Atg14L (e) and vice versa (f). WCL, whole cell lysate; IP, immunoprecipitated. See Supplementary Information, Fig. S6 for full scans of blots in a, d, e and f.
ErbB4 but not ErbB4 kinase activity mediates Nrg3 functions in synaptogenesis.Figure 4 A) His6-tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations. Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho-tyrosine (pY) and ErbB4, and of whole lysates using antibodies against pErk1/2 and pAkt   A) His6-tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations. Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho-tyrosine (pY) and ErbB4, and of whole lysates using antibodies against pErk1/2 and pAkt   C, D) Neurons from Nrg3-/-;ErbB4-/-;vGAT-Cre mice were transduced with a lentivirus expressing either (C) ErbB4wt or (D) ErbB4kd (kinase dead ErbB4) in a Cre-dependent manner, and with a second virus at a low titer expressing Nrg3/SypCFP. Immunocytochemical analysis of ErbB4 and SypCFP; (C,C') and (D,D') each show the same image, (C',D') show the SypCFP signal   E-G) Quantification of Synapse preference towards ErbB4+ neurons (E), ErbB4 enrichment (F) and SypCFP enrichment (G) in synapses on neurons expressing wildtype ErbB4 (wt) or ErbB4kd (kd)   H) Nrg3-/- neuron cultures were transfected with a plasmid that strongly overexpresses Nrg3 and GFP and immunostained with antibodies against ErbB4, GFP and GluA; (H) shows the triple stained image, while (H') and (H'') only show ErbB4 and GluA signals, respectively   I) Co-cultures of Nrg3-/- neurons with HEK293 cells expressing Nrg3. Cultures were stained with antibodies against Nrg3, ErbB4 and GluA. Note that Nrg3, ErbB4 and GluA are enriched at the contact sites between Nrg3-expressing HEK293 cells and ErbB4+ neurons   J) Schematic diagram of the strategy used to obtain paired recordings in cultures obtained from Nrg3-/-;Gad1/Gad67GFP mice. Inhibitory neurons were identified by GFP expression. The cultures were transduced with lentiviruses expressing either Nrg3 or Nrg3∆EGF (N3 and N3∆, respectively) together with nuclear red fluorescence protein (nRFP). Transduced neurons, identified by nuclear RFP fluorescence, were stimulated and evoked responses were recorded from interneurons that were GFP-positive and nRFP-negative at 12-15 days in culture  K-M) Sample traces of paired recordings (K), quantification of the amplitude of the evoked excitatory postsynaptic currents (eEPSC) (L) and quantification of paired pulse ratios (M)
Figure 61×106 CD14+ myeloid cells from healthy donors were polarized by 1 ng/ml GM-CSF in the presence of BSA-control (C), 0.2 mM oleate (O) or 0.2 mM stearate (S) over a 6-day period. The expression of CD206, CD204 and CD38 was analyzed via flow cytometry. The level of lipid droplets was determined via BODIPY staining. The oxygen consumption rate (OCR) of differentiated myeloid cells was monitored as described in Materials and Methods. (D) The purified CFSE-labeled autologous CD4+ T cells were co-cultured with polarized myeloid cells with indicated treatment for T cell proliferation assay. Tumor tissue (tumor) as well as corresponding non-tumorous adjacent tissue (control) were collected from colorectal cancer patients and prepared for histopathology. The expression of CD68 (red), CD206 (green) and ADRP (blue) indicate the lipid droplets in tumor infiltrating myeloid cells. Nuclei (white) were stained using DAPI. The absolute number of positive cells was quantified in 5 high-power fields (hpf) (scale bar = 20 µm).
Figure 1Overview of the study populations (cohorts) and schematic proteomic workflow. The CSF of three cohorts comprising AD and control subjects were analyzed. The total number of subjects per cohort group is depicted. Light and dark shades represent female and male subjects, respectively. 'Ctrl' refers to non-AD control subjects. Number of proteins identified and quantified passing the 1% FDR cutoffs in each sample. Horizontal lines show the mean and the error bars ± SD. The dashed line indicates the level of the meta-median (1233 proteins) of the group medians of quantified proteins. Number of samples per group as shown in A). Data completeness curve. The number of proteins in the dataset (Y axis) depending on the minimum number of samples in which the proteins have each been quantified (X axis) is plotted. The arrows indicate 50%, 75%, and 100% data completeness. Median CSF protein abundance distribution as calculated from MS-intensities of quantified peptides of each protein. The top ten most abundant proteins and hemoglobin proteins are highlighted. Global correlation map of proteins generated by clustering the Pearson correlation coefficients of all possible protein combinations. The abundance of proteins with common regulation correlate across samples and they therefore form a cluster. Prominent clusters are annotated with functional terms obtained from bioinformatics enrichment analysis. The position of tau (gene name MAPT) is labeled on the Y axis. The inset shows the color code for Pearson correlation coefficients.
– Mutants of cdkb1 but not cdka;1 are hypersensitive to cisplatin.Figure 4A. The wildtype and CDKA;1-DE mutants were grown on control plates for 10 days. Root lengths were measured 10 days after germination.B. The wildtype and CDKA;1-DE mutants were grown on plates containing 1 mM hydroxy urea for 10 days. The mutant wee1 was used as a positive control for hydroxy urea sensitivity. Root lengths were measured 10 days after germination.C. The wildtype and CDKA;1-DE mutants were grown on plates containing 0,6 μg/mL bleomycin for 10 days. The mutant ku70 was used as a positive control for bleomycin sensitivity. Root lengths were measured 10 days after germination.D. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on control plates for 10 days. Root lengths were measured 10 days after germination.E. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on plates containing 1 mM hydroxy urea for 10 days The mutant wee1 was used as a positive control for hydroxy urea sensitivity. Root lengths were measured 10 days after germination.F. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on plates containing 0,6 μg/mL bleomycin for 10 days. The mutant ku70 was used as a positive control for bleomycin sensitivity. Root lengths were measured 10 days after germination. G. The wildtype, CDKA;1-DE (grey bar) and the double mutant cdkb1;1cdkb1;2(purple bar) were grown on control plates and were transferred to plates containing 15 μM or 30 μM cisplatin three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs.H. Graphs represent the ratio of the mean growth rate on 15 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype, CDKA;1-DE and cdkb1;1cdkb1;2.I. Graphs represent the ratio of the mean growth rate on 30 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype, CDKA;1-DE and cdkb1;1cdkb1;2. J-N. Images show a wild-type plant, CDKA;1-DE and the double mutant cdkb1;1 cdkb1;2 (from left to right) on the indicated day after germination and the indicated drug treatment.
Figure 3HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3, ASC or pro-casapase-1 (Pro-Casp-1). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose. BMDMs were treated with indicated stimuli. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody. BMDMs were treated with indicated stimuli. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody. BMDMs were treated with indicated stimuli. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody. BMDMs were treated with indicated stimuli. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody. A schematic representation of NLRP3 wild type and deletion mutants. HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3 or NLRP3 deletion mutants as indicated in (F). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose. A schematic representation of ABRO1 wild type and deletion mutants. HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3 and Flag-tagged ABRO1 or ABRO1 deletion mutants as indicated in (H). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.
Figure 8Figure 8. Model showing distinct gene regulatory function of JNK signaling during EMT. Onset of EMT crucially relies on Smad-mediated (Valcourt et al, 2007; Xu et al, 2009) transcriptional changes and as cells advance through the mesenchymal fate, JNK pathway progressively becomes critical in gene regulation. We identified a set of transcription factors (Prkrir, Etv6, Srf, Hoxb9, Tsc22d1, Tcf12, Nr1h4, Zeb1, Pcbp4, Nfil3) that are induced by JNK signaling and function in progression and/or maintenance of the mesenchymal state Furthermore, JNK activity also represses another set of factors (Meox2, Hnf4a, Sfpi1, Lmo7, Cdx2) that promote an epithelial phenotype. Among these JNK regulated factors, in addition to the factors known to regulate EMT and carcinogenesis (Etv6 (Tognon et al, 2011), Srf (Bae et al, 2014), Hoxb9 (Hayashida et al, 2010), Tcf12 (Lee et al, 2012), Zeb1 (Burk et al, 2008; Sanchez-Tillo et al, 2013), Meox2 (Valcourt et al, 2007), Hnf4a (Santangelo et al, 2011), Sfpi1 (Rosenbauer et al, 2006; Walter et al, 2005), Lmo7 (Tanaka-Okamoto et al, 2009) and Cdx2 (Gross et al, 2008)), we also identified five novel transcription factors that are crucial for progression and/or maintenance of EMT (Nfil3, Nr1h4, Tsc22d1, Pcbp4 and Prkrir).
Proposed model of RNAlow neoblast regulation.Figure 7At homeostasis, RNAlow neoblasts are relatively slow-cycling and their size is restricted by Lrig-1. During regeneration, RNAlow neoblasts grow, extend projections, and enter the cell cycle by 48 hpa in a TORC1-dependent manner.
Figure 4Ribbon diagram and locally sharpened surface representation (transparent= of the three TssJ protomers in orange, labelled TssJ.i, TssJ.o and TssJ' for the inner, outer and additional monomer respectively. In light green and blue are the TssM.i and TssM.o protomers respectively Superimposition of the ribbon diagrams of TssM-TssJ heterodimer within the inner (green-orange) and the outer (blue-orange) pillars Ribbon diagram of the TssM.o (outer pillar) with the TssJ.o and the TssJ' (orange) Fluorescence of prey cells (green) indicating their survival against attacker cells after 4 h. The image of a representative bacterial competition spot is shown on the upper part. The relative fluorescent level (in AU) and the number of recovered E. coli recipient cells (in log10 of colony forming units (cfu)) are indicated in the lower graphs. The assays were performed from at least three independent cultures, with technical triplicates and a representative technical triplicate is shown. Error bars in the left graph represent ± standard deviation (SD) from technical triplicate. The circles in the right graph indicate values from technical triplicate, and the average is indicated by the bar. Time-lapse fluorescence microscopy recordings showing localization and dynamics of the sfGFPTssM fusion protein in the parental (WT) and TssJ mutated strains (TssJ D97K). Individual images were taken every 60 seconds. The corresponding time of each micrograph is indicated in the lower right part of the image. Stable foci in the WT strain and less stable foci in the TssJ D97K strain are respectively indicated by white and blue arrowheads. Scale bars, 1 µm.
Fig.Figure 1(A) Gross morphology of control (Ctrl), Sas-4−/−, Sas-4−/− 53bp1−/−, and Sas-4−/− Usp28−/− embryos at E9.5. At least five embryos were considered per genotype and all the double mutant embryos exhibited the rescue criteria mentioned in the text. Scale bar = 500 μm. (B, C) Immunostaining for p53 (B) and Cleaved-Caspase3 (Cl-CASP3, C) on transverse sections of Ctrl, Sas-4−/−, Sas-4−/− 53bp1−/−, and Sas-4−/− Usp28−/− embryos at E9.5. The area shown encompasses the ventral neural tube and surrounding mesenchyme. Asterisks in (B) denote non-specific staining of blood cells. Scale bar = 50 μm. (D, E) Quantifications of the p53 (D) and Cl-CASP3 (E) fluorescence signals shown in (B) and (C), respectively. Three embryos per genotype were used for the quantifications. *** p < 0.001, *p<0.05 (two-tailed student's T-test, one-way ANOVA with Tukey's multiple comparisons tests gave similar results). Bars represent mean ± s.d. (D) Ctrl: 1.00 ± 0.06 (n=1770 cells); Sas-4−/−: 6.84 ± 0.34 (n=275), Sas-4−/− 53bp1−/−: 1.85 ± 0.75 (n=690), Sas-4−/− Usp28−/−: 1.50 ± 0.48 (n=975). (E) Ctrl: 1.00 ± 0.70 (n=1690); Sas-4−/−: 7.88 ± 2.62 (n=175); Sas-4−/− 53bp1−/−: 1.43 ± 0.71 (n=1030); Sas-4−/− Usp28−/−: 1.57 ± 0.44 (n=950).
Figure 2A Representative pellets of WT and mutant G1E-ER-GATA-1 cell clones treated with or without b-estradiol for 48 hours. B Wright-Giemsa staining of untreated and b-estradiol-treated (48 hours) WT clone 1 (WT1) as well as double-mutant clone 1 (int1/8 Mut1). C Growth curves in WT1 and double mutant clone 1 cells treated with or without b-estradiol (n = 3, mean +/- SE). D Western blotting of ER-GATA-1 and a-tubulin in WT and mutant clones. E Real-time RT-PCR of Alas2 mRNA in untreated or b-estradiol-treated WT and mutant clones. ***, P < 0.001 with respect to all values of WT1-3 (n = 4, mean +/- standard error (SE)) (left panel). Alas2 mRNA in intron 1 mutant clones was compared to that in intron 1/8 double-mutant clones. ***, P < 0.001 with respect to all values of intron 1 mutants 1 and 2 (right panel). F Quantitative ChIP analysis of ER-GATA-1 occupancy at the GATA sites of Alas2 introns, HS2 of the b-globin locus control region, and the Hbb-b1 promoter in WT clone 1 and mutant clones. Alas2 exon2/intron2 junction site was used as a negative control. ***, P < 0.001 with respect to WT1 (n = 4, mean +/- SE).
ACE2 expression is upregulated in human airway epithelial cells in response to rhinovirus infection.Figure 6. ACE2 expression is upregulated in human airway epithelial cells in response to rhinovirus infection.Primary epithelial cells from 4 donors were exposed to rhinovirus or medium only (used as control). 24 hrs post-infection, gene expression was assessed using Affymetrix U133 plus 2.0 human GeneChips. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA137767; GEO: GSE27973).