EMBO/sd-character-level-ner huggingface dataset:
Figure 2A Schematic of three chamber test. B Representative animal tracks in the sociability test. C Percentage of time the subject mice spent in each chamber in the sociability session of three-chamber test. Mock (n=11) stranger 1 (S1) chamber vs. empty (E) chamber; ZIKV (n=16) stranger 1 chamber vs. empty chamber; one-way ANOVA with Bonferroni post hoc test. D Quantification of time spent investigating the stranger mouse and empty cage during sociability session. Mock (n=11) sniffing time at stranger 1 mouse vs. empty cup, ZIKV (n=16) sniffing time at stranger 1 mouse vs. empty cup. E Preference indices in sociability. Mock (n=11) vs. ZIKV (n=16). F Representative animal tracks in the social memory test. G Percentage of time the subject mice spent in each in the social memory session. Mock (n=11) stranger 1 (S1) chamber vs. stranger 2 (S2) chamber, p< 0.0001; ZIKV (n=16) stranger 1 chamber vs. stranger 2 chamber; one-way ANOVA with Bonferroni post hoc test. H Quantification of time spent investigating the familiar mouse and novel stranger mouse during social novelty session. Mock (n=11) sniffing time at stranger 1 vs. stranger 2 mouse; ZIKV (n=16) sniffing time at stranger 1 vs. stranger 2 mouse. I Preference indices in the social novelty session of the three-chamber test. Mock (n=11) vs. ZIKV (n=16). Data information: All data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed unpaired t-test unless described otherwise; n represent biological replicates. J Schematics of five-trial social memory test. K Sniffing time in each trial of the five-trial social memory test. Mock (n=11) trial 5 vs. trial 6; ZIKV (n=16) trial 5 vs. trial 6. L Normalized sniffing time in each trial of the five-trial social memory test. Mock (n=11) trial 5 vs. trial 6; ZIKV (n=16) trial 5 vs. trial 6. M Schematics of the self-grooming test. N Time spent grooming in the self-grooming test. Mock (n=11) vs. ZIKV (n=16). Data information: All data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed unpaired t-test unless described otherwise; n represent biological replicates.
Figure 3(A) Volcano plot (log10) of the most enriched/decreased proteins in the mitochondrial proteome of pex3Δ versus pex3Δmsp1Δ strains as detected by quantitative mass spectrometry (n=3, biological replicates, n=1, technical replicate). 2010). Effect sizes were calculated for Cohen's d A protein's relevance to the model was determined using a p-value threshold of < 0.05 with an absolute effect size threshold of 1 or greater (large effect). Gray dots represent all measured proteins. Red dots highlight peroxisomal-associated proteins. (B) Heatmap (log2) of peroxin protein levels from pex3Δ and pex3Δmsp1Δ as detected by quantitative mass spectrometry (n=3). Protein classes are indicated in the row labels. Values were z-score normalized by protein. (C) Mitochondria from the indicated yeast strains (wild type, pex19Δ, pex19Δmsp1Δ), expressing Pex13-V5 (under its endogenous promotor) were either left untreated (lane 1,2,3) or exposed to Na2CO3 and separated into the membrane pellet (4,5,6) or supernatant fraction (7,8,9). After separation by SDS-PAGE the fractions were immunoblotted for Pex13-V5 (α-V5) and porin (α-porin).
Experimental workflow and the phagosome proteome of M2 (IL-4) macrophages.Figure 2(A) Workflow of the phagosome proteomics experiment. (B) Volcano plot of the phagosome proteome data. 1,766 proteins were quantified of which 121 proteins were significantly up-regulated and 62 proteins were down-regulated in M2(IL-4) macrophages. Selected proteins are indicated. (C) Heatmap of proteomics data shows high reproducibility between biological replicates. Selected proteins are highlighted. (D) Selected Gene Ontology (GO)-terms of biological processes significantly up-regulated on phagosomes of M2(IL-4) macrophages. (D) Selected Gene Ontology (GO)-terms of biological processes significantly down-regulated on phagosomes of M2(IL-4) macrophages.
Figure 2A, B Real-time PCR (qRT-PCR) analyses of WTAP mRNA expression in PBMCs (A) or BMDMs (B) upon challenge with VSV-eGFP, poly(I:C), HSV-1 or HSV60 at indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. C qRT-PCR analyses of WTAP mRNA expression in A549 cells upon challenge with VSV-eGFP, poly(I:C), HSV-1 or poly (dA:dT) at the indicated time points. D qRT-PCR analyses of WTAP mRNA expression in A549 cells stimulated with IFN-α or -β at the indicated time points. E A549 cells were stimulated with poly (I:C) for 16 hrs and then treated with PR-171 (10 μM) or PS-341 (10 μM) for additional 4 hrs and 8 hrs. The cell lysates were analyzed by immunoblotting. Representative images from 3 independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right). F A549 cells were stimulated with poly (I:C) for 16 hrs and then treated with CQ (50 mM) or Baf A1 (0.2 mM) for additional 4 hrs and 8 hrs. The cell lysates were analyzed by immunoblotting. LC3B served as a good marker of the autophagy process. Representative images from 3 independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right). G Immunoassays of extracts of A549 cells infected with VSV-GFP for 16 hrs and then treated with PR-171 (10 μM) or DMSO for additional 4 and 8 hrs, followed by immunoprecipitation with anti-WTAP antibody and immunoblot analysis with anti-Ub antibody. H Coimmunoprecipitation and immunoblot analyses of extracts of 293T cells transfected with various combinations of plasmids encoding FLAG-tagged WTAP, and HA-tagged K48-linked, K63-linked or wild-type ubiquitin and treated with DMSO or PR171 (10 μM).
Figure 7A. AT-MSCs were pretreated with a TGFβRI inhibitor SB431542 at 10 µM, or control DMSO for 1 h, and subsequently exposed to r.h. TGF-β1 at indicated concentrations. The cells were cultured for another 24 h and harvested for miR-21 assay. The expression of miR-21 was normalized on RNU6B. Data is given as mean ± SEM of three independent experiments. B-D. AT-MSCs were transfected with 30 nM Smad7-siRNAs or control siRNA. Untransfected AT-MSCs served as control. RNA and protein were isolated 2 days after transfection for Smad7 expression by qPCR (B) and western blot (C). The remaining cells were treated with r.h. TGF-β1 at indicated concentrations for another 24 h. The expression of human TGF-β1 was analyzed by qPCR and normalized on human GAPDH (D). Data is given as mean ± SEM of three independent experiments. E-G. AT-MSCs were transfected with 5 nM miR-21 mimic or control mimic, and were exposed to r.h. TGF-β1 2 days after transfection at indicated concentrations for another 24 h. The expression miR-21 was analyzed by qPCR-based miR-21 assay normalized on RNU6B (E), Samd7 protein level was shown by western blot (F), and human TGF-β1 expression was monitored by qPCR normalized on human GAPDH (G). Data is given as mean ± SEM of three independent experiments. H-J. AT-MSCs were transfected with 50 nM miR-21 inhibitor or control inhibitor, and were exposed to r.h. TGF-β1 2 days after transfection at indicated concentrations for another 24 h, and subsequently subjected to the analysis of expression of miR-21 (H), Smad7 protein (I) and human TGF-β1 mRNA (J). Data is given as mean ± SEM of three independent experiments. K-L. Full-thickness excisional wounds were produced on CD18-/- mice by 6 mm biopsy punches. One day after wounding, each wound received intradermal injection of 2.5x105 AT-MSCs that had been transfected with 50 nM miR-21 inhibitor or control inhibitor 2 days after transfection. CD18-/- wounds received mock injection with PBS served as control. Each wound region was digitally photographed at days 0, 3, and 6 post-wounding, and expressed as percentage of the initial wound size at day 0 (K). Wound tissues were harvested on day 6 post-wounding for quantification of total TGF-β1 protein by ELISA (L). Data is given as mean ± SEM; n = 5 wounds per group; *, p<0.05; **, p<0.01, by one-way ANOVA with Tukey's test. MSC_miR-21-IN, miR-21 inhibitor transfected AT-MSCs; MSC_ctrl-IN, control inhibitor transfected AT-MSCs.
Frequently concurrent loss of NANOG, LKB1 and NKX2-1 expression in human IMA.Figure 3(A) Representative images of low, medium, high protein expression of NANOG in human lung adenocarcinomas. Scale bar, 150 μm (top), 50 μm (bottom). (B) Representative H&E and IHC staining for NANOG in human ADC and IMA. Scale bar, 50 μm. (C) Statistic analysis of NANOG expression in ADC and IMA. The tumor tissues were analyzed by IHC staining and graded as low, medium, high expression. Significance was calculated by χ2 test for trend. (D) Western blot for NANOG and TUBULIN in 7 paired human normal lungs (N) and ADC samples (T) and human ESCs. Mr, relative molecular mass. kDa, killoDalton. (E) Western blot for NANOG and TUBULIN in human adenocarcinoma cell lines transfected with empty vector or human NANOG overexpression vector. The arrow indicated the band of exogenous human NANOG. The asterisk indicated the band of endogenous human NANOG. Mr, relative molecular mass. kDa, killoDalton. (F) Representative images of low, medium, high protein expression of LKB1 in human lung adenocarcinomas. Scale bar, 150 μm (top), 50 μm (bottom). (G) Representative H&E and IHC staining for LKB1 in human ADC and IMA. Scale bar, 50 μm. (H) Statistic analysis of LKB1 expression in ADC and IMA. The tumor tissues were analyzed by IHC staining and graded as low, medium, high expression. Significance was calculated by χ2 test for trend. (I) The expression grade of IHC staining of NANOG, NKX2-1, LKB1 in 76 human IMA (1 sample/row).
Figure 8A schematic illustration of kinome siRNA library screening for identifying synthetic lethal targets in resistant WM3248 cells.B B A graph plotting the Z scores of normalized cell viability after siRNA transfection (two replicates). siRNA targets with Z scores lesser than -2 in both replicates were considered as synthetic lethal hits. C qRT-PCR analyses confirming knockdown of TESK1 mRNAs in resistant WM3248 cells after transfection of two distinct TESK1 siRNAs for 48 hr. D Cell viability analyses of parental and resistant cells. Cells were transfected with the indicated siRNAs for 6 days. WM3248 cells were cultured in media containing either 2 % or 10 % FBS before cell viability assay. Relative cell viability was measured by CCK8 assay. E Fluorescence micrographs showing YAP/TAZ and actin filaments in resistant WM3248 cells. Cells were transfected with the indicated siRNAs for 72 hr before staining. F Quantification of YAP/TAZ localization in the experiment presented in (E). G qRT-PCR analyses of the expression of YAP/TAZ-target genes (ANKRD1, CTGF, and CYR61) and TESK1. Resistant SKMEL28 and WM3248 cells were transfected with either control siRNA or TESK1 siRNA pool (1+2) for 48 hr.
Bosutinib degrades lentiviral Aβ1–42 in a parkin-dependent mannerStaining of 20μm brain sections shows intracellular Aβ1–42 within the (A) hippocampus of lentiviral Aβ1–42 injected WT mice, and (B) Bosutinib clearance of intracellular Aβ1–42.Figure 4Staining of 20μm brain sections shows intracellular Aβ1-42 within the (A) hippocampus of lentiviralAβ1-42 injected WT mice, and (B) Bosutinib clearance of intracellular Aβ1-42. Staining of 20μm brain sections shows intracellular Aβ1-42 within the (C) cortex of WT mice with the lentiviralAβ1-42, and (D) Bosutinib clearance of intracellular Aβ1-42 (N = 7). ELISA of human soluble and insoluble Aβ1-42 at 6 weeks post-injection of lentiviralAβ1-42 and daily treatment with 5 mg/kg Bosutinib (N = 9) for 3 weeks. * Significantly different to control or as indicated, Mean ± SEM, ANOVA with Neumann Keuls multiple comparison. WB on 4-12% NuPAGE SDS gel of total brain lysates in 1 year old wild type and parkin−/−mice treated with 5 mg/kg Bosutinib for 3 weeks on 4-12% NuPAGE SDS gel (Invitrogen) showing parkin (1st blot) total Abl (2nd blot), T412 Abl (3rd blot), Beclin-1 (4th blot) and LC3 (5th blot) relative to actin (N = 7). Staining of 20μm brain sections shows plaque formation with 6E10 antibody and DAB 6 weeks post-injection with (G) lentiviralAβ1-42 + 3 weeks DMSO treatment and (H) IP injection with 5 mg/kg Bosutinib 3 weeks post-lentiviral expression (3 weeks treatment) clears plaques in WT mice (N = 7).(I) Lentiviral Aβ1-42 + DMSO and (J) IP injection with 5 mg/kg Bosutinib 3 weeks post-lentiviral expression (3 weeks treatment) in parkin−/−mice (N = 7).
Figure 11A. Schematics illustrate the soluble Bax monomer and the MOM-bound Bax monomers, dimers and tetramers. The helices α2-α9 are depicted as cylinders with the indicated colors, and the helix α1 is omitted for simplicity. The MOM-bound tBid, depicted as a star, enhances cytosolic Bax interaction with the membrane (step I) and binds to the groove of Bax, thereby releasing α9 from the groove to the membrane (step II). The tBid binding also unfolds the Bax separating the latch (α6-α8) from the core (α2-α5) (step III). After tBid dissociates, the activated Bax monomer either embeds more hydrophobic regions such as α5 and α6 into the membrane (step IV) or binds other activated Bax monomer forming a BH3-in-groove dimer on the membrane surface (step V). After α5 is released into the membrane, the remnant BH3-in-groove dimer is rearranged to a α2-α3-α4 dimer (step VI). A Bax tetramer is then formed by two BH3-in-groove dimers via an additional dimer interface formed by two membrane-embedded α9 helices either intersected (step VII) or in parallel (step VIII). These Bax complexes may be in equilibrium with one another, but the BH3-in-groove dimerization is a prerequisite for the other dimerizations. In principle, combinations of these dimer interfaces would be sufficient to generate higher order oligomers, although other dimer interfaces have been discovered.B. A lipidic pore in the MOM is illustrated with BH3-in-groove dimers located on the cytosolic surface of the membrane and α9 monomer and dimer at the rim. Note that the BH3-in-groove dimers are partially embedded in the cytosolic monolayer, generating membrane tension that induces the pore, while the α9 monomer and dimer are integrated into the highly curved monolayer to reduce the line tension, thereby stabilizing and expanding the pore.
NLRP3 is required for inflammasome activation downstream of TRIM21 (A,B,C) Primed HMDM were treated with MCC950 (1 μM) for 30 mins before stimulation overnight with AdV (50 000 pp/cell) and h9C12 (20 μg/ml) or IVIg (20 mg/ml) or 10 μM Nigericin.Figure 6Primed HMDM were treated with MCC950 (1 μM) for 30 mins before stimulation overnight with AdV (50 000 pp/cell) and h9C12 (20 μg/ml) or IVIg (20 mg/ml) or 10 μM Nigericin. IL-1β (A) or TNF (B) or LDH release (C) were measured in cell supernatants. Data shows average ± s.d. of two independent donors and is representative of three independent experiments. WT, NLRP3, ASC or Caspase-1 - deficient THP-1s were stimulated as in (A) and IL-1β in the supernatant measured by ELISA. Data is representative of two independent experiments. HMDM were primed with 10 ug/ml pI:C for 2h, treated with 100 μM VX-765 for 30 mins before stimulation for 16 h as in (A). IL-1β or LDH release was measured in cell supernatants. Data shows average ± s.d. of two independent donors Primed HMDM were treated with KCl as indicated for 1h, before being stimulated for a further 6h as in (A). IL-1β and TNF were measured in cell supernatants by ELISA. Data is representative of three independent donors. Primed HMDM were stimulated with AdV and 20 μg/ml h9C12 or 10 μM Nigericin for 3h, and then immunostained for ASC or intracellular antibody. Co-localisation was assessed by confocal microscopy. Data is representative of two independent donors.
Figure 8A. Continuous osmotic delivery of miR-10b inhibitor markedly reduces tumor burden. The osmotic pumps, loaded with lipid nanoparticles formulated with 2'-O-MOE-POmiR-10b inhibitor or non-targeting control infused 2 μg of the oligonucleotides per day introtumorally, over 13 days. Tumors growth was monitored by the WBI, and the left panels illustrate tumor imaging of representative animals at the end of treatment. The bars represent average signal ratios for each group at day 13 (end of treatment), relative to day 2 (treatment initiation). B. Representative immunostaining of tumors for PCNA proliferation marker. C. Representative immunostaining of the tumors for KI67 proliferation marker. D. Representative tumor immunostaining for cleaved Caspase 3 as a marker of apoptosis. E. Tumor cell invasion was examined by fluorescence microscopy for mCherry-positive cells migrating through the tumor border. F. Quantitative immunostainings analysis indicates that proliferation and apoptosis markers are affected by anti-miR-10b treatment. No significant effect on invasion of intracranial GBM8 was observed.
Depletion of ubiquilin inhibits the degradation of autophagosomes by lysosomes.Figure 5(A) Autophagosome subcellular localization during starvation was analysed by microscopy in UBQLN‐ or autophagy‐related gene ATG5‐KD cells expressing GFP‐tagged microtubule associated protein 1 light chain 3 (GFP‐LC3). (B) Autophagosome spatial distribution was quantified by measuring the distance of each LC3 vesicle from the nucleus (in pixels) and by analysing the frequency distribution. (C) Control or UBQLN‐KD cells expressing GFP‐LC3 were starved and stained with the mitochondria dye Mitotracker™. Dual staining of LC3 (green) with Mitotracker (magenta) was analysed by microscopy. (D) Control and UBQLN‐KD cells stained with Mitotracker were starved for 6 h; BAF A was added where indicated. Mitotracker fluorescence was measured by flow cytometry. Unless specified otherwise, values for UBQLN‐KD and control+BAF A were compared with that of controls at the same time points, with ***P0.001, **P0.01 and *P0.05 (paired Student's t‐test). (E) Cells were transfected with pShooter™ together with control or UBQLN (1+2) siRNA. Expression of pShooter was comparable in control and UBQLN‐KD cells (top). Cells were starved for 3 or 6 h, and the intensity of GFP was quantified by flow cytometry (bottom). The fluorescence value obtained with controls (no starvation) was considered as 100%, and the value obtained for the 3‐ and 6‐h time points was normalized to controls. The graph represents the mean±s.e. of four assessments. (F) Cells expressing a chimaeric LC3 fused to both GFP and mCherry were transfected with control or UBQLN siRNA, and starvation was induced for the indicated times. Puncta from 25 cells were scored and analysed by microscopy for their fluorescence colour. The graph represents the mean of three experiments±s.e. The number of mCherry single‐positive puncta in UBQLN‐KD cells was compared with that of controls for each time point; **P0.01 and *P0.05 (paired Student's t‐test). (G) Control or UBQLN‐KD cells were starved for increasing periods. Where indicated, BAF A was added to the starvation medium (+B). Cell lysates were analysed by Western blot for their content in LC3‐II. Loading control: transferrin receptor (TfR). BAF A, bafilomycin A1; GFP, green fluorescent protein; KD, knockdown; siRNA, short interfering RNA; UBQLN, ubiquilin.
Glucose and glutamine promote GOT2 acetylation and GOT2–MDH2 association A, B Glucose and glutamine increase GOT2 K159 acetylation and GOT2–MDH2 association in HEK293T cells.Figure 2A, B Glucose and glutamine increase GOT2 K159 acetylation and GOT2-MDH2 association in HEK293T cells. Flag‐GOT2 and Myc‐MDH2 were overexpressed in cells treated with increased concentrations of glucose (A) or glutamine (B) for 6 h. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with Myc‐MDH2 were determined by Western blot analysis. C, D Glucose and glutamine increase GOT2 K159 acetylation in Panc‐1 cells. Flag‐GOT2 was overexpressed in cells treated with different concentrations of glucose (C) and glutamine (D) for 4 h. GOT2proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis. Relative GOT2 K159 acetylation levels were normalized against Flagprotein levels. E, F Quantification of the percentage of K159‐acetylated endogenous GOT2 in Panc‐1 cells. Recombinant fully K159‐acetylated GOT2 was loaded onto the same gel, together with endogenous GOT2 from Panc‐1 cells treated without or with glucose (12 mM) (E) or glutamine (2 mM) (F) for 4 h. GOT2 protein and K159 acetylation were detected by Western blot. The percentages of K159 acetylation in GOT2 were calculated after normalizing against GOT2 protein levels. G, H 3K mutant GOT2 displays negligible response in changing protein association with MDH2 after glucose or glutamine treatment. Panc‐1 cells with GOT2 knockdown and re‐expression of wild‐type or 3K mutant GOT2 were treated with glucose (G) or glutamine (H) at the indicated concentrations for 4 h. The protein association between Flag‐tagged wild‐type or 3K mutant GOT2 and endogenous MDH2 was determined by Western blot analysis.
Figure 2(A-D) Generation of Golgi stacks by AmphoB. Stacked Golgicisternae are observed in quick-frozen replicas (A-C) and substituted thin section (D) of atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml) for the indicated time. (A) Scale bar = 1 µm. (B) The magnified image shows the area indicated by the square in (A). Scale bar = 0.5 µm. (C, D) A representative Golgi stack with four cisternae is shown. Scale bar = 0.5 µm. (E-G) Generation of AP-like double-membrane compartment from the stacked Golgi. A quick-frozen replica (E) and a substituted thin section (F, G) are shown. Scale bars = 0.5 µm. The distal stacks are extended, curved, and form AP-like structure (E, F). Mitochondria are encircled by the Golgi membrane (F). (G) The AP-like structure was separated away from Golgi stacks. (H, I) The scene that AP-like structure was fused with vacuole. A magnified image of (H) is shown in (I). In atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr), outer membrane of AP-like structure (red arrows) was fused with vacuolar membrane (blue arrows). Inner membranes of AP-like structure (yellow arrowheads) were entered into the vacuoles to become AB-like structure. Green arrows indicate the membrane fusion site. Scale bars = 0.5 µm (H) and 0.2 µm (I). (J) The number of elongated single cisternae (green bars), Golgi stacks (red bars), and AB-like structures (blue bars) was counted over time using electron microscopy images. Error bars indicate s.e.m. (3 hr: n = 21, 6 hr: n = 20, 12 hr: n = 193, 24 hr: n = 192). (K) Translocation of the trans-Golgi protein to the vacuolar membrane induced by AmphoB treatment. GFP-Sft2-expressing atg5Δ cells (trans-Golgi marker protein) were incubated with or without AmphoB (2.5 µg/ml, 24 hr), and localization of GFP-Sft2 was assessed by confocal microscopy. Arrowhead indicates the GFP signal on the vacuolar membrane. Scale bars = 2 µm. (L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (L, M) Representative electron microscopy images of grh1Δatg5Δpep4Δ (L) and gvp36Δatg5Δpep4Δ cells (M) treated with AmphoB (2.5 µg/ml, 24 hr). AP-like and AB-like structures were absent. (L) A unilamellar Golgi cisterna was observed in the cytoplasm (dotted square). Scale bar = 1 µm. (M) Formation of a Golgi stack, but not Golgi curvature, was observed (dotted square). Bar = 0.5 µm. (L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (N) Indicated cells were treated with AmphoB for 24 hr and the number of cells containing AB-like structures was counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells. (L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (O) GFP-pho8Δ60-expressing atg5Δ, grh1Δatg5Δ and gvp36Δatg5Δ cells were cultured with or without AmphoB (2.5 µg/ml, 24 hr) and subjected to western blotting for GFP. An unidentified band at 32kD (asterisk) is non-specific band because it was present in Pep4-lacking grh1Δatg5Δ cells.