EMBO/sd-character-level-ner huggingface dataset:
Molecular profile of astrocytes under pro- and anti-neurogenic conditions.Figure 6A tSNE plot of cluster analysis revealed the presence of all expected DG cell types identified by known markers. B DG cell types were present in all three experimental conditions (young non-runner in red; young runner in dark blue; aged in light blue). Box indicates the cluster containing exclusively DG astrocytes. C tSNE plot representing astrocytes in all three experimental conditions. D Comparison of astrocytes derived from the DG of young non-running animals to those of young runners revealed no differential clustering between the two conditions. E Statistical overrepresentation test by Panther; selected Gene ontology (GO)-terms for biological processes are represented for genes downregulated (red) and upregulated (dark blue) in astrocytes from running mice. F Comparing astrocytes from the DG of young control animals to those of aged mice did not result in differential clustering. G Panther statistical overrepresentation test for selected GO-terms for biological processes for genes downregulated (red) and upregulated (light blue) in astrocytes derived from aged animals.
Figure 7(A-B) Representative fluorescence microscopy images of wild type (A) or NimB4sk2 (B) macrophages expressing Rab5 (middle panel) or Rab7 (right panel) driven by Hml∆-Gal4 and stained with the LysoTracker Red (live imaging). Overlay of fluorescence and DIC. Scale bar = 10 μm. (C) Quantification of the mean fluorescence intensity of LysoTracker in macrophages from larvae (confocal live imaging). Values from at least three independent experiments are represented as mean ± SD (** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).
βHBP fails to induce rapid oligomerization of TIFA A) IL-8 production after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S.Figure 2A) IL-8 production after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysate in HeLa cells (Hoechst in blue, IL-8 in red) after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysate in HeLa cell B) Quantification of IL-8 a with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat C) TIFA oligomerization at 6 h in TIFA-GFP-expressing HeLa cells treate with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat at 6 h in TIFA-GFP-expressing HeLa cells treate D) Quantification of TIFA oligomerizatio with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat E) TIFA oligomerization at 30 min in cells treate F) Kinetics of TIFA-GFP oligomerization upon treatment with digitonin alone or in combination with βHBP or S. flexneri lysate
a) Confocal micrographs of HeLa cells expressing yellow fluorescent protein (YFP) fused to Nap1, Sintbad or Tank and infected with S.figf1(a) Confocal micrographs of HeLa cells expressing yellow fluorescent protein (YFP) fused to Nap1, Sintbad or Tank and infected with S. Typhimurium, stained for ubiquitin (Ub) 2 h after infection. At least 100 cells were analyzed. DAPI, 4,6-diamidino-2-phenylindole. Scale bar, 10 μm; arrowheads, bacteria shown in insets. (b-e) Normalized ratio between luciferase activity bound to beads and present in lysates. The indicated purified GST fusion proteins coupled to beads were incubated with lysates of 293ET cells expressing Nap1, Sintbad or Tank, each fused to luciferase (b) (c), lysates of 293ET cells expressing the indicated Nap1 mutants fused to luciferase (d), lysates of E. coli expressing Nap1(1-85) fused to luciferase (e) lysates of 293ET cells expressing Nap1, NEMO or Optineurin fused to luciferase
Figure 5A PLX4032 dose-response curves of parental cells expressing wild-type YAP or YAP-5SA. Mock-transfected cells were used as a control. B Immunoblotting for the indicated proteins. Lysates of mock, wild-type YAP and YAP-5SA expressing cells were analyzed. Cells were treated either with PLX4032 (2 &amp;amp;#x3BC;M) or DMSO for 24 hr. C qRT-PCR analyses of YAP target genes (ANKRD1, CTGF and CYR61), MITF and SOX10 in wild-type YAP or YAP-5SA expressing melanoma cells compared with mock transfected cells.All
Induction of PGCLCs from human iPSCsSchematic presentation of PGC‐precursor and PGCLC induction.Figure 2Schematic presentation of PGC‐precursor and PGCLC induction. FACS analysis of the concentration‐dependent effect of BMP4 on TRA‐1‐81+/cKIT+ PGCLCs on day 4. B25: 25 ng/ml BMP4; B50: 50 ng/ml BMP4; B100: 100 ng/ml BMP4. FACS analysis of TRA‐1‐81 and cKIT during PGC induction of up to day 8. Gene expression analysis of selected PGC markers in TRA‐1‐81+/cKIT+ and SSEA1+/cKIT+ FACS fractions of d4 PGCLCs. Samples were calibrated with iPSC values, and iPSC values depict 1.
Figure 6Diagram of in vivo timeline (D = day). i.t. administration (20µM) of Tat-P4-(C5)2 significantly reduced ipsilateral paw hypersensitivity of SNI mice in the initiation stage (2 days after injury) of the spared nerve injury model at time points 1 and 3 hours (n = 7) i.t. administration (20µM) of Tat-C5 did not affect ipsilateral paw hypersensitivity of SNI mice in the initiation stage (2 days after injury) of the spared nerve injury model (n = 8) Representative western blots (top) for GluA1 and GluA2 following surface biotinylation of spinal cord slices. Densitometric analysis (bottom) of immunoblots. We observed up-regulation of both GluA1 and GluA2 surface level following SNI surgery compared to non-operated animal CTL (unpaired t test GluA1: t(4)=3.372, GluA2: t(4)=3.991,#p≤0.05, n=3/group). Tat-P4-(C5)2 but not Tat-C5 significantly reduced the SNI-induced GluA2 surface expression and shows a strong tendency for GluA1 as well. Representative immunoblot shows unaltered PICK1 total level in the total lysate in the four conditions. Images of lumbar spinal cord sagittal sections showing the time course of TMR-Tat-P4-(C5)2 (magenta) after i.t. administration (20 µM) in SNI mice showing maximal accumulation in cells after 60 min. Scale bar 1000 µm and 100 µm in Zooms. Overlay with immunohistochemical staining for the neuronal marker NeuN (yellow) highlights strong neuronal tropism of TMR-Tat-P4-(C5)2 (magenta). Scale bar 100 µm Dose-dependent Von Frey test in the acute phase (2 days after injury) of the spared nerve injury model following i.t. administration of Tat-P4-(C5)2 (n=8/group) did not affect ipsilateral paw hypersensitivity of SNI mice and lasts up to 4h post injection. i.t. administration of Tat-P4-(C5)2 (20 µM) at day 14 and 28 after injury in the maintenance phase of SNI model in mice shows significant increase in pain withdrawal threshold up to 3 hours with equal efficacy in male and female mice (compared to time 0 pre-injection, D14 n=8/group/gender, D28 n=6 male, n=7 female).
Figure 5(A) TOPFlash reporter activity following differentiation for 3 days in 3 µM CH or 100 ng/ml (W100) or 200 ng/ml (W200) recombinant Wnt3a plus 500 ng/ml recombinant RSpondin1. Relative luciferase units are normalised to FOPflash. Average and SD of three independent experiments. (B) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR at day 2 of differentiation in the presence or absence of 3 µM CH or recombinant Wnt3a (200 ng/ml plus 500 ng/ml RSpondin1(WR)). Average and SD of three independent experiments. ** p<0.01. (C) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR in Ctnnb1 null cells at day 1 and 3 of differentiation in the absence or presence of 3 µM CH. Average and SD of three independent experiments. (D) Immunoblot for Tcf7l1 in Ctnnb1 null cells during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH. Results from two independent, clonally-derived Ctnnb1 null ES cell lines are shown. Tubulin was used as a loading control.
Figure 3(A). Amino acid sequences encoding for the enzyme activity and RNA binding ability in E.coli TruB and human TruB1 (Top). Design of mutant 1 (mt1) and mutant 2 (mt2) (bottom). (B). In vitro enzyme activity assay. 32p-UTP-labelled tRNAphe were treated with recombinant TruB1, mt1, or mt2. The strong upper bands represent uridine (U), and the lower weaker bands represent pseudouridine (Ψ) on autoradiographs of the TLC plate. (C). Relative miRNA expression of let-7a in HEK-293 cells infected with tetracycline-inducible expressing lentiviruses for TruB1, mt1, mt2 or GFP 5 days after doxycycline treatment, as determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD. (D). Northern blotting for let-7a and RNU6B in HeLa cells infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP, 5 days after doxycycline treatment. (E). Hybridization intensities of (D) were quantified and normalized to ctrl (GFP). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD. (F). Pseudouridylation activity of TruB1 for tRNA and pri-miRNAs. 32p-UTP-labelled tRNAphe, pri-let-7a1 or pri-miR-10a were treated with recombinant TruB1. Upper bands represent uridine (U), lower bands represent pseudouridine (Ψ) in autoradiographs of the TLC plate. (G). Location of pseudouridine sites detected by the CMC primer extension method. Total RNA purified from HEK-293FT cells were treated with CMC. CMC treated RNA were reverse-transcribed with RI-labelled specific primers for tRNAphe or pri-let-7b. ddATP was used for sequence control. Pseudouridines are indicated by black arrows.
Short-term, but not long-term, proteasome disruption triggers autophagic clearance of aggregates.Fig 4(A-D) Inhibition of the proteasome by overexpression of a temperature sensitive proteasome mutant (DTS). Dmef2-Gal4, UAS-GFP-Atg8a control and UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a and animals were shifted to the restrictive temperature then starved and CQ treated 6 hrs prior to dissection. 18 hrs post-shift (PS), both control (A) and DTS muscles (B) accumulate GFP-Atg8a punctae, but the latter also accumulate ubiquitin (Ubi) aggregates. 48 hrs PS, control muscles (C) maintain strong autophagy induction, but DTS mutant muscles (D) have reduced GFP-Atg8a punctae accumulation, similar to the Rpn1 knockdown phenotype. (E) Quantification of the mean GFP-Atg8a punctae and Mander's overlap coefficient for GFP-Atg8a and Ubi for genotypes shown in A-D (SEM is indicated for n = 8 ventral longitudinal muscles from individual animals with p-values of DTS relative to control). Dark green = Dmef2-Gal4, UAS-GFP-Atg8a; Light green = UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a. (F) Western blot analysis of endogenous Atg8a from third instar muscle lysate. 6 hrs starvation induces lipidated Atg8a-II in Dmef2-Gal4 control at both 18 hrs and 48 hrs PS, but not in DTS at 48 hrs PS.
Figure 4A. Representative images of dressed bodies of the adult male and CtM Hanwoo (n = 10 in each group). B. Dressed body weight, thickness of the dorsal subcutaneous fat, total weight of the extramuscular fat (posterior subcutaneous fat, mesenteric fat, and retroperitoneal fat), and serum levels of the branched-chain amino acids (BCAAs). C. Representative images of fresh striploin muscle from the adult male and CtM carcasses (n = 5 in each group). D. The intramuscular fat area in size-normalized muscle. E. The intramuscular metabolome profiles analyzed using GC-TOF-MS and clustered by PCoA based on the Bray-Curtis dissimilarity matrix F, G. The relative abundances of intramuscular metabolites (F) and β-hydroxybutyrate (3-HB) (G) in the male and CtM samples. H. The serum and mucle ketone body levels, quantified enzymatically, as described in the Materials and Methods section (n = 10 in each group).
Figure 2A AP was induced by 5 injections (IP) of cerulein at a dosage of 50 μg/kg. SAP was induced by AP and one injection of lipopolysaccharide (LPS, IP) at a dosage of 20 mg/kg. AP+ANGPTL4 (2 mg/kg) was induced by AP and one injection of ANGPTL4 (IP) at a dosage of 2 mg/kg. ANGPTL4 (4 mg/kg) was induced by two injections of ANGPTL4 (IP) at a dosage of 2 mg/kg. Mice were sacrificed 6 h after pancreatitis induction. (H&E and immunofluorescent staining for IL-1β (red) and amylase (green) was performed along with immunohistochemistry for ANGPTL4 in pancreas tissues (n = 10, each group). Scale bar represents 50 µm B Concentrations of the inflammatory cytokines TGF-β, IL-1β, and IFN-γ as well as ANGPTL4 in the sera (n = 10, each group). C TUNEL staining for apoptosis and vacuoles formation (TEM). Red arrow, apoptotic cells; white dotted line, vacuoles region. D T.E.M. ANGPTL4 immunogold labelling were performed in pancreas tissues from the AP, SAP and ANGPTL4-treated models (n = 5, each group).
a) Expression of Ulk1 and Atg5–12 in the indicated erythroid cells.f1(a) Expression of Ulk1 and Atg5-12 in the indicated erythroid cells. (b-e) Induction of conventional macroautophagy in WT and Ulk1gt/gt, but not in Atg5−/−, embryonic erythroid cells. Ter119+ erythroid cells from WT, Atg5−/−, and Ulk1gt/gt embryonic mice (E18.5) were treated with 1 μM rapamycin (b,c) and 1 μM STS (d,e), and then harvested at the indicated times. (b,d) Representative protein expression of LC3 and p62 measured by western blot. Actin was a loading control. Uncropped images are shown in Supplementary Fig. 10. (c,e) Semi-quantitative analysis of LC3-II/LC3-I and p62 protein expression (n=3, mean±s.d.). Asterisks indicate a significant difference at P0.05 (analysis of variance (ANOVA)). #P0.05 versus value of WT 24 h (ANOVA). 'NS' indicates not significant (ANOVA). (b-e) Induction of conventional macroautophagy in WT and Ulk1gt/gt, but not in Atg5−/−, embryonic erythroid cells. Ter119+ erythroid cells from WT, Atg5−/−, and Ulk1gt/gt embryonic mice (E18.5) were treated with 1 μM rapamycin (b,c) and 1 μM STS (d,e), and then harvested at the indicated times. (b,d) Representative protein expression of LC3 and p62 measured by western blot. Actin was a loading control. Uncropped images are shown in Supplementary Fig. 10. (c,e) Semi-quantitative analysis of LC3-II/LC3-I and p62 protein expression (n=3, mean±s.d.). Asterisks indicate a significant difference at P0.05 (analysis of variance (ANOVA)). #P0.05 versus value of WT 24 h (ANOVA). 'NS' indicates not significant (ANOVA).
UBE4B and LSD1 double knockdown activates both proteasomes and autophagy.Fig 5(A) Increased protein levels of proteasome subunits upon the UBE4B and LSD1 double knockdown in HEK293T cells . (B) The UBE4B and LSD1 single or double knockdowns increase the proteasomal degradation of a reporter substrate, Suc-LLVY-Luciferin, whose degradation is measured in a luciferase release assay. The double knockdown shows synergistic proteasomal activation when compared with the individual knockdowns (n = 6). (C) The autophagy activity is significantly increased in cells with the UBE4B and LSD1 double knockdown. The in vivo activity of the ATG4B protease, which cleaves LC3 precursors, was quantified via a Gaussia luciferase release assay (see S5D Fig., S5E Fig., and Materials and Methods for details). The HCT116 cells were analyzed 48 h after the initiation of the knockdown. (D) Quantification of LC3-II levels in HEK293T cells with the UBE4B and LSD1 double knockdown or the mock control. The cells were treated with or without 10 mM 3-methyladenine (3-MA) for 48h before lysis. Western blots indicate an increase in LC3 levels (top panels) in the double UBE4B and LSD1 knockdown cells (middle panels), while the actin control is unchanged (bottom panels). The graph shows the quantification of LC3-II levels as normalized to actin levels (n = 3). A one-way ANOVA test with matched multiple comparisons and Tukey correction was used for statistics. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Fig.Figure 9Evidence for the dependence of UapA translocation to the PM on Sec24, Sec13, actin and SsoA tSNARE, as well as, for the need for actin polymerization in COPII formation, is described in the text. The role of actin for the hypothesized close-range transport and fusion of uncoated vesicles carrying UapA to the PM is highly speculative, as well as the existence of an ER-to-PM intermediate compartment. For details see main text.
i.Figure 6Accumulated chow intake (normalized by body weight) measured during 7 days following a single i.c.v. injection of vehicle or 10 pmol AβOs in mice (n = 13 Veh; 10 AβOs; data are representative of two independent experiments with similar results). ***P < 0.0001; Student's t-test. Daily body weight measured during 7 days after i.c.v. injection of vehicle or AβOs (n = 7 animals/group; data are representative of two independent experiments with similar results). Adult Swiss mice received a single i.c.v. injection of vehicle or 10 pmol AβOs, and hypothalamic levels of mRNA for AgRP (C; n = 6 Veh; 5 AβOs), NPY (D; n = 6 Veh; 5 AβOs) and POMC (E; n = 7 animals/group) were analyzed 7 days after injection. In (C), *P = 0.0191; in (D), *P = 0.0115; Student's t-test. Swiss mice received a single i.c.v. injection of vehicle (Veh) or 10 pmol AβOs, and their brains were analyzed by Fluorojade staining of degenerating cells 7 days after the injection. Representative images of Fluorojade staining in the hypothalamus of vehicle- or AβO-injected mice (n = 4/group). Scale bar = 100 μm in left panels (top and bottom) and 20 μm in right panels (top and bottom). Positive control (bottom left panel) was the hippocampus of a mouse that received one i.c.v. injection of quinolinic acid (36.8 nmol) and was analyzed 24 h after.
– Transcriptome as well as gain and loss of function analyses implicate TLR8 as bacteria and mitochondria ribosomal RNA sensor.Figure 2A Cytokine release of 3 days differentiated (ddi) THP1 cells toward stimulation with TLR ligands and bacterial RNAs (S, Svedberg; r, ribosomal; tot, total; n = 3). B, C Responsiveness of Unc93b1-/--3ddiTHP1 cells treated with T2.5 and challenged with heat inactivated (hi) bacteria (triangle, decreasing doses; n=3). D Ratios of constitutive mRNA amounts in 3- versus 8ddi (grey bar) and of each of both versus undifferentiated THP1 cells (black or white bar, respectively) according to a transcriptome profiling result (n = 1). E, F NF-κB driven relative luciferase activity (Rel. luc. act.) of hTLR8 overexpressing HEK293 and murine RAW264.7 cells, respectively, upon challenges (vector, empty plasmid; n = 3). G Transfection of TLR8 siRNA impairs 3ddiTHP1 cell responsiveness (n = 3).
Western blot analysis of siRNA-mediated knockdown of selected MaMTH-HTS interactors on CFTR glycosylation in CFBE cells.Figure 5A) Blot images showing bands B and C as well as the calnexin internal control. B) Blot quantification showing relative fold change of band B and band C versus treatment with a non-targeting siRNA (Neg1). One-way Anova followed by Dunnett's post hoc test was performed to determine significance versus baseline (siNeg1 + DMSO treatment, *p<0.05) and 5 µM VX-661 (#p<0.05). Measurements were obtained from biological triplicates (n=3) and all values are mean ± s.d.
Figure 3A-B. Oxidized mitochondria with the radioactive single- or double-cysteine Bax proteins, or the single-cysteine Bax protein pairs were prepared and analyzed as in Figure 1B. C. Oxidized mitochondria with the radioactive double-cysteine Bax protein were prepared, subjected to Na2CO3 extraction and analyzed as in Figure 1D. D. The best structural model for the helices α2-α3-α4 dimer is shown with the residue pairs replaced by cysteine pairs in (B) presented as in Figure 1A. The β carbon atoms of each residue pair are linked by a dashed line with the distance ranging from 4.7 to 5.2 Å. E. The structural model for the α2-α3-α4 dimer is shown with the residues that are buried in the MOM or exposed to the aqueous milieu as determined in Figure 2 presented in yellow or cyan stick form, respectively. The pdb file for the model is provided as Dataset EV1 and named as "Helices a2-a3-a4 dimer.pdb".
Figure 6A Macrophage cells were treated with ANGPTL4 (100 ng/ml) for 24 h and then cytokine levels were analyzed by a mouse cytokine profiler assay (n=3). Mean pixel density was quantified using ImageJ software analysis. B ANGPTL4 and C5a expression levels were measured in bone marrow-derived macrophages from pancreatitis-induced ANGPTL4-/- and WT mice. C Phospho-kinase array was performed to find the mechanism by which ANGPTL4 increased C5a in human THP1-derived macrophage cells (n=3). Cells were treated with ANGPTL4 (100 ng/ml) for 6 h. After phospho-kinase array analysis, the expression of AMPKα, JNK, AKT, P70S6K, and EGFR was confirmed by western blotting. D To explore whether ANGPTL4 affects JNK, AMPK, and PI3K/AKT signaling, each inhibitor (SP600125, compound C, and HS-173, 1 µM) was treated to in THP1-derived macrophages for 2 h after ANGPTL4 treatment (100 ng/ml) for 6 h. E C5a, C5aR, TNF-α, IL-6, and IL-1β mRNA levels were identified by qPCR in mouse macrophages or primary acinar cells after treatment with ANGPTL4 and LPS (100 ng/ml). In the transwell experiment, mouse primary acinar cells were cultured in the lower well and macrophages were incubated in the upper transwell bucket treated with ANGPTL4 (100 ng/ml). Expression of ANGPTL4, C5aR, and C5a was determined in siANGPTL4-treated THP1-derived macrophage after LPS activation. F In the transwell experiment, TUNEL positive cells and C5aR expression were identified in primary acinar cells after C5aR antagonist (W-54011, 10 nM) pretreatment for 12 h prior to treatment with ANGPTL4, LPS, and C5a (100 ng/ml) for 24 h to macrophage in transwell. Scale bar represents 20 µm
facilitates aerobic glycolysis and proliferation A, Schematic diagram of the mRNA structures of the glycolytic genes and the indicated STAT5 binding site.Figure 5A, Schematic diagram of the mRNA structures of the glycolytic genes and the indicated STAT5 binding site. B, phosphorylation of STAT5 under INSL5 stimulation. CNE1 cells were starved in FBS-free medium for 24h and then treated with INSL5 peptide (50ng/mL) in the presence of an anti-INSL5 antibody at 100ug/ml, or an isotype-matched IgG control (IgG) for 30min. C, Localization of STAT5. A confocal analysis of STAT5 staining was performed using CNE1 cells transfected vector or INSL5. Scale bars represent 20μm D, Cytoplasmic and nuclear proteins from vector or INSL5overexpressing cells were separated and subjected to immunoblot as indicated. The nuclear marker Lamin B1 and the cytoplasmic marker GAPDH were used to demonstrate the purity of fractions. E, INSL5 overexpression activated AKT, ERK, JAK1 and STAT5 phosphorylation, not STAT3 in HK1 cells. INSL5 overexpressing HK1 cells were treated with MK2206 (50uM), U0126(20uM), MK2206+U0126, or Ruxolitinib (5uM) for 24h. Western blotting was performed to evaluate the effects of those inhibitors on STAT5 and STAT3 phosphorylation level. F, ChIP-qPCR analyzed the occupancy of potential E-boxes by STAT5 in the genes of HK2, Glut3 and PFK1 in INSL5-overexpressing CNE1 cells using IgG or anti-STAT5 antibodies. G, Luciferase activity of different glycolytic gene promoter reporters in 293T cells transfected with STAT5 or empty vector. H, Glucose uptake assessed by 2-NBDG incubation in INSL5 overexpressed CNE1 cells with STAT5 knock down. I, Glucose uptake was assessed by 2-BNDG incubation in INSL5 overexpressed HK1 cells treated with JAK kinase inhibitor (Ruxolitinib). J, MTT assay of vector control or INSL5 overexpressing HK1 cells either treated with STAT5 siRNAs or JAK kinase inhibitor.