EMBO/sd-character-level-ner huggingface dataset:
(f) In vitro-translated Flag-Parkin was incubated with bacterially expressed GST or GST fused to the Fbxo7 Ubl domain (1-88) immobilized on glutathione beads. Bead-bound proteins and inputs were analyzed by immunoblotting with anti-Parkin antibodies.
Western blot analysis of Flag-RNF8 denaturing-IP in HeLa cells showing RNF8 hyper-ubiquitination in ∆ATX3 condition. RNF8 hyper-ubiquitination was suppressed by reintroduction of GFP-ATX3-WT but not with GFP-ATX3-C14A or GFP-ATX3-UIM*.
C,D: Superimposition of the structure of SARS-CoV-2 N-NTD (light blue) with MERS-CoV N-NTD dimer (grey) or MERS-CoV N-NTD bound to P3 (cyan) (PDB: 6KL6) (Lin et al, 2020). The detailed interactions are shown in D.
Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. GFP chimerism in KLS (I) and KLS- (J) cells in the BM of secondary recipients (mean + individual values, Kruskal-Wallis test with Dunn's post-test, NaCl: n = 7, G-CSF: n = 6, CoPP: n = 8 mice per group).
d, Representative flow cytometry data of CD69 and CTV staining on gated living CD8 T cells. The purified CD8 Tn were isolated from 45-wk-old mice and stimulated by different doses of anti-CD3 ab for 72 hours. Enlarged number representing the percentage of the corresponding gate.
(J) Quantification and representative images of CD206+ cells over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bars: 50 µm.
TDP-43 intensity in TDP-43/NeuN double-positive cells in CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition; 5 sections per mouse). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.
D) Representative images of Control (GFP-tagged 1-70 of Tom70), GFPMiro1 and GFPMiro2 in WT MEFs stained with catalase (peroxisomes).
(G) The p-values assigned by GENDULF to genes within chr11p12-p13, chr6p21.3, chr3q29 and chrXq22-q23 chromosomal segments, ordered by their location. The lower dashed line represents a significance threshold corrected for the number of genes evaluated and the upper dashed line represents a significance threshold corrected for all genes and transcripts in GTEx, with alpha = 0.05. Data information: ** is P-value<0.01 and *** is P-value<0.001, using the permutation test The P-values in panels G are for the hyper-geometric enrichment test.
B. In vitro modification reactions containing the six fragments and HeLa NE were performed. After nuclease P1 digestion, the products were analyzed by 1D-TLC with pU and pψ as controls.
The proteins corresponding to the mRNAs in each group were clustered according to their expression profiles. Differential protein expression was defined as fold change > 1.15 and FDR < 0.2 (t test, Benjamini-Hochberg corrected) based on two independent experiments. The mean expression of each cluster is shown in black.
J. Mia40 overexpression reduces the toxicity of polyQ proteins. In newly made cells, Q97-GFP is still toxic (though less than in wild type) but after several generations, cells escape sensitivity by loss of Rnq1 nucleation seeds.
I. Poly(I:C) and LPS increase mTOR P-S2159 in primary bone marrow-derived macrophages (BMDMs): BMDMs were stimulated -/+ poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min).
J. mRNA levels of the puckered transcripts from retinas of untreated or AD4 treated wild type and ADAM17-/- mutant retinas. n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.
(A) Responses of Col-0 wild type (WT), atg5, atg7 and nbr1 plants to Botrytis. Wild-type and mutant plants were inoculated by spraying with spore suspension at a density of 2.5×105 spores ml−1, and kept at high humidity. Pictures of representative plants were taken at 4 and 5 days post inoculation (dpi). (B) Quantitative real-time PCR analysis of the B. cinerea ActA (BcActA) transcript levels in infected Arabidopsis plants at 5 dpi. The experiments were repeated twice with similar results.
d) Previously hypoxic cells, frequently undergo apoptosis after reoxygenation. We noted an accumulation of intensely orange fluorescing spots at the edges of compact UnaG positive areas. Scale bar = 50µm.
A-C Viability counts of the indicated V. parahaemolyticus (A-B) or E. coli (C) prey strains before (0 h) and after (4 h) co-incubation with the indicated V. parahaemolyticus attackers on media containing 3% NaCl at 30°C. In A and B, media also contain 0.1% arabinose to induce expression from plasmids. In A, prey strains contain either an empty plasmid (pEmpty) or a plasmid for arabinose-inducible expression of VP1389 (pVP1389). In B and C, prey strains contain an empty plasmid that provides a selection marker, and the attackers are derivatives of a ∆hns mutant (parental). In B, the attackers contain an empty plasmid, or plasmids for the arabinose-inducible expression of VP1388 (pVP1388) or VP1390 (pVP1390). Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. DL, assay detection limit. ∆hcp1 was used as a T6SS1- control strain. The experiment was performed at least three times with similar results; results of a representative experiment are shown.
L. Carfilzomib activates NF-κB signaling pathway through IRE1α in M2 macrophages. Wild-type and Ern1-/- Raw264.7 cells were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by Carfilzomib (1 μM) for indicated time points. WCL were analyzed by IB with indicated antibodies.
E. The localization of COPI•Dsl BiFC foci compared to the autophagy marker Ape1p. Double fluorescence micrographs of the BiFC pair β'-COPVN•Dsl3pVC and the autophagy marker mRFPApe1p are superimposed with the DIC image. BiFC-YFP signals are pseudocolored green. Fluorescent signals appeared distinct from each other. Also no co-localization was observed when we analyzed COPI•COPII(Sec16p) BiFC spots in cells expressing mRFP tagged APE1. Scale bar 10 µm.
A. Targeted deletion of nucleolin GAR domain by CRISPR-Cas9. Schematic shows mouse nucleolin exons targeted by single guide RNAs (sgRNAs) and resulting deletion in the GAR domain amino acid sequence.
H, Differential expression of EIF4G1 and ASNS in high and low THEM6 tumours according to the PRAD TCGA dataset. Data information: Centre line corresponds to median of data, top and bottom of box correspond to 90th and 10th percentile, respectively. Whiskers extend to adjacent values (minimum and maximum data points not considered outliers). Statistical analysis: two-tailed Mann-Whitney U test. Data reproducibility: : n = 225 tumours for THEM6 low and n = 121 tumours for THEM6 high.
(C) Western blot analysis in lung lysates from vehicle and barasertib treated mice using Col1α and Fn1 antibodies. Gapdh is used as loading control. *P< 0.05, **P<0.005, ***P < 0.0005, 1-way ANOVA, (n=4-5 mice/group).
(I) atg13 cells expressing GFP‐Atg8 and containing wild‐type Atg13, the Atg13‐FV mutant or an empty plasmid were grown to mid log phase with and without starvation for 4 h in SD‐N. Processing of endogenous Ape1 and cleavage of GFP‐Atg8 was analysed by western blotting, and quantified by calculating the ratio of cleaved versus uncleaved Ape1 or GFP‐Atg8, respectively. The asterisk marks a non‐specific band detected by the Atg13 antibody. Figure source data can be found with the Supplementary data.
B) Activation of FcγRs IIB/C and IIIA by patient serum is mediated by serum derived sICs. Patient serum samples were depleted of sICs by PEG precipitation and the supernatant (SN) was compared to untreated serum regarding FcγR activation (left). Shown is one exemplary experiment performed in technical duplicates. Error bars indicate reproducibility as range. IgG concentration in the precipitate (PC), supernatant (SN) or unfractionated serum respectively is shown in the bar graph (right).
H. HEK293 cells were treated for 8 or 24 h with 100 μM H2O2 or, alternatively, for 2 h with 2 mM hydroxyurea (HU), selected as positive control of CHK1 phosphorylation at Ser317. Phospho-CHK1Ser317 was assessed by Western blot; densitometry normalized to Vinculin, selected as loading control, and expressed as arbitrary units (with HU arbitrarily set to 1). Values shown represent the means ± SD of n = 3 independent experiments. n.s., not significant.
A) Metabolic profiles of HeLa cells in dependence on IF1 levels under glycolytic and OXPHOS conditions generated by an automatic flux analyzer measuring oxygen consumption rates and extracellular acidification rates. n=3 independent experiments (error bars = SD).
(h) Colony formation assay showing that stable overexpression of SLC7A11 reversed Sorafenib-induced cell death in YAP/TAZ-deficient HCC cells. HLE-shLuc and HLE-shY/T were or were not transfected to overexpress SLC7A11 (SLC7A11 OE) and treated with DMSO or 6μM Sorafenib for 2 weeks as indicated. Results represent three independent experiments.
H Quantification of KP∆NC tumour cell-derived spheres after adenoviralGFP‐tagged p53 overexpression.
Quantification of specific proteins band intensities from immunoblot in panel B using a CCD camera. Intensities of protein bands were normalized to intensity of actin protein band.
IB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG-β-catenin and Myc-HRAS-WT, -2A, or -2E for 32 hr
(L) A proposed model showing how Merlin is activated to induce Lats1 phosphorylation on the plasma membrane.
(B-C) 25HC depletes accessible cholesterol on the plasma membrane. (B) Calu-3 cells were treated with indicated concentrations of 25HC for 1h, and subsequently incubated with ALOD4 for 30 min. Cell were washed twice with PBS, lysed, and the amounts of bound ALOD4 were examined by western blotting. (C) Calu-3 cells were treated with 5 µM 25HC for 1h, and subsequently incubated with AF488 labeled ALOD4 for 30 min. Cells were washed twice and examined by fluorescence microscopy.
Quantification of the experiments shown in (C). Shown is the quantification of the GFP signal at the mCherry-LacR-Fok1 focus.
(A) Proliferation assay of Stat3+/+ and Stat3-/- cells cultured in N2B27-based 2i media either in the presence or absence of LIF. Cells were seeded and scored for four consecutive days. Scores were normalized to day1. Mean and s.d. of three technical replicates. See also Appendix Figure S1A.
E Cytoplasmic accumulation of the soluble cell-wall precursor UMP in vancomycin-treated (blue line), AMSIN-treated (red dashed line) or not treated (green line) culture of MRSA P1374. Inset: UMP identified by UPLC-HRMS with singlely and doubly protonated m/z observed.
HDAC6 KO MEF cells are defective in aggregate clearance but not in autophagosome induction or targeting. (A) Filter‐trap analysis of MG132‐induced, SDS‐insoluble ubiquitinated aggregates generated in wild‐type (WT), HDAC6 KO, and KO MEFs reconstituted with different HDAC6 constructs as indicated. The signal intensity from the ubiquitinimmunoblot (bottom panel) was quantified and presented as the average of the means from three independent experiments along with the standard deviation (top). Note the significant accumulation of ubiquitin‐positive aggregates in HDAC6 KO MEFs and KO MEFs stably expressing HDAC6‐CD and ΔBUZ mutants. WT, HDAC6 KO, and HDAC6 KO MEFs stably expressing human HDAC6 (hHDAC6 WT), HDAC6 CD (catalytically inactive mutant), or HDAC6 ΔBUZ (ubiquitin‐binding‐deficient mutant) were analysed for the level of HDAC6 using an anti‐human HDAC6 antibody. Mouse endogenous HDAC6 (mHDAC6) and actin levels were determined by each corresponding antibody.
A. Deconvolved inverted fluorescent micrographs of BiFC signal of Chm7-VC with either Heh1-VN or Heh2-VN. Cell borders are outlined. Scale bar is 5 μm.
RNA-seq was conducted on day-3 of differentiation induction of Tnfaip2-/- EBs that were either treated with PA/PC or with a vehicle control (n=3 independent pools of 7-62 EBs per group). The heat maps depict the intersection of DEGs with the following gene sets (G) "ectoderm development". Five other stem cell-related genes (Tfcp2l1, Dppa3, Fbxo15, Zfp42 and Klf2) were additionally incorporated based on literature searches. The asterisks indicate the reverted changes in gene expression that were seen in Tnfaip2-/- EBs versus WT EBs The color scale represents the gene-wise z-score calculated from normalized gene expression levels.
(H) GAPDH-MHRhiv expression in CHME3 reduces infection with HIV-1-WT-luc. Statistical significance was determined by t test. * = p<0.05. Data are mean values from three independent experiments +/- SEM. Molecular weight markers (in kDa) are shown to the right of WBs.
(A) Real-time qPCR analysis of ANXA1 expression using mRNA extracted from the non-cancerous SB, cancerous 10C, and human CRC cell lines HCT116, DLD1 and RKO, each grown to 50% or 100% confluency. All results were normalized to the ANXA1 mRNA levels in SB cells of 100% confluency, which was designated as 1. Data are mean values ± SEM. The experiment was performed in triplicates and repeated twice. **p<0.01 and ***p<0.001 (student's t-test).
(D) Pharyngeal pumps/minute in young adult gas-1 mutant nematodes treated with vehicle or methylxanthines. Error bars: mean ± SEM, n=18. ****p value< 0.0001, one-way ANOVA, Dunnett's post-hoc correction.
Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. Heatmap of non-targeted metabolites in atosPBG compared to wild-type embryos shown with average log2fold change (FC) in the non-targeted LC-MS/MS analysis (F) We observe increases in atosPBG in intermediates of mitochondrial fatty acid β-oxidation (FAO), including different carnitine-conjugated lipids, and (F-G) a significant increase in ketone body substituents compared to the control. Data information: Values in heat maps are obtained from untargeted metabolomic analysis, unpaired t-test
C RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains (either ter1+ or ter1Δ) cultured in YES medium for ~ 43 pds. Values represent polyA+ TERRA detected in immunoprecipitated material expressed as a fraction of the input (graph on the left) or after normalization to ACT1 RNA in the corresponding input (on the right), and expressed as fold increase over an untagged wt strain (unt ter1+). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to unt ter1+; two-tailed Student's t test).
B) Co-immunoprecipitation analysis to elucidate the interaction between Suv39h1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Suv39h1 or 3xFlag-ΔN-Suv39h1 were subjected to immunoprecipitation with the anti-Flag tag antibody followed by immunoblotting with the indicated antibodies. IgG represents the heavy or light chain
Immunofluorescence images (F) and quantification of the percentage of ciliated cells (G, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (G), >150 cells were analyzed for each experiment. Scale bar, 5 µm.
(E) MSA plot of DPPA2, DPPA4 and DPPA4ΔSAP ChIP-seq in mESCs showing enrichment over L1Md_A, L1Md_T and MERVL-int sequences. Gaps are in grey and sequences in white with overlap of color-coded ChIP-seq signals. Upper plot shows average coverage of the signal over the aligned TEs.
A) HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) and grown in fed (FED) or starvation conditions (STV) in the presence or absence of CLQ for 1h. Cells were then fixed and stained using LC3B-II (red) antibody. Analysis of LC3B puncta number is reported (bottom). All data are expressed as the mean value ± SEM. Statistical analysis were performed by two-way ANOVA followed by Tukey's multiple comparison test. ****, P<0.0001. (n=4 independent experiments, n≥25 fields analysed). Scale bar 5 μm.
MCF-10A cells incubated in the absence or presence of puromycin (200μM) were fractionated on sucrose gradients, and distribution of CIP2A-BP transcripts was quantified by qPCR.
B. Representative segment of an electron micrograph of negatively stained Elongator after stabilization by gradient fixation (Lane 6 of Fig 1A). Circled particles correspond to intact Elongator. White scale bar corresponds to 50 nm.
(B) Total protein extracts from MPH (left panel) or mouse liver (right panel) were subjected to Western blot with an antibody against NFIL3. ACTB was used as loading control.
(E) Whole body temperature before (pre), one hour (1 hr Post), and 24 hours (24 hrs Post) after intraperitoneal injection of corn oil, vehicle (corn oil and saline), 0.1, 0.5, and, 1 mg BAM15 per kg of body weight (N=11 per group),
Labeling for CD31 in retinas of P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice after daily i.p. injection of 200 µg of 4OHT from P12. Scale bar, 500 µm. Quantification of areas of neovascular tufts in P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice. n = 6 per group.
(B) WASH knockout accelerates LC3 conversion by confocal microscopy. Endogenous LC3 puncta were visualized by staining with antibody against LC3 (left panel), and calculated as shown in the right panel. Nuclei were stained with DAPI. Scale bar, 10 μm.
IKBKE gene (encoding for IKKε) was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. 16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. IKKε CRISPR-Cas9 knockout MCF10A cells are less invasive compared CRISPR Control (nontargeting crRNA) cells. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.
0.0001.D) Schematic of in vitro tube formation assay; F-domain scaffolds (designed scaffolds) were added at 90 nM F-domain and removed at 24h; vascular stability was analyzed at 48h and 72h timepoints.
A Illustration of the target points of the used pharmacological and protein-based inhibitors
c, REST cKO mice show age-related neurodegeneration. Shown are sections of the hippocampal CA1 subfield from 8-month-old REST cKO mice labelled for TUNEL, cleaved caspase 3 (cl-CASP3) and GFAP, or stained with haematoxylin and eosin (HE).
E. Effect of oligo#5 on RNA1/RNA2 ratios in the injected area. The area marked by Cy3 staining in the dorsal hypothalamus, consisting mainly of the arcuate nucleus was microdissected, RNA isolated and analyzed by qPCR. NS: non-significant changes (3 hrs: p=0.01; 6 hrs: p=0.000001; 9 hrs: p=0.02; 12 hrs: p=0.06; n=3)
IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h, followed by a 30 min pulse with either vehicle control (Vh), 5 mM ATP or 20 µM Nigericin. Data shown are pooled of 5 independent replicates.
C Phenotypic analysis of lymphocyte and monocyte subpopulations in peripheral venous blood from T2D COVID-19 patients treated in Gen or requiring ICU treatment.
(A) FM images of pex3 atg1 cells grown for 16 h on MM-M/G. Cells produced Pex14-mCherry and C-terminal mGFP fusions of the indicated proteins.
F. Representative immunofluorescence images and quantification of IFT88 levels at the centrosome and primary cilium. RPE1 cells were transfected with control or PCM1 siRNA#1 for 48 h, serum starved for 24 h and treated with DMSO or 0.5 μM MLN8237. Cells were fixed and immunostained with antibodies against IFT88, acetylated tubulin and gamma tubulin. DNA was stained with DAPI. Scale bar, 10 μm. G. Quantification of IFT88 centrosomal intensity and ciliary concentration for F. IFT88 fluorescence intensities at the centrosome and axoneme were measured from maximum projections, and average means of the levels in control cells were normalized to 1. Ciliary concentration was calculated by dividing ciliary fluorescence intensity to the cilium length. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; ****p < 0.0001, Unpaired Student's t-test)
F. Representative FACS readout of tetramethylrhodamine ethyl ester (TMRE) stained cells upon 100 nM GSK-LSD1 or DMSO treatment after 6 days. G. Quantification of tetramethylrhodamine ethyl ester (TMRE) stained cells upon 100 nM GSK-LSD1 or DMSO treatment at indicated time points.
(B) Western blot for CLU, Cdc25C-T48, Cdk1 and PP2A in PC3 cells after siSCR or siCLU transfection and after synchronization followed by treatment with or without okadaic acid (OA) (100 nM) for 12 hours. Proteins were extracted 15 min after nocodazole release. Vinculin was used as loading control.
D, Diagram showing the 462-nt sequence in length from NoV∆B2 RNA1 was inserted into the 3' untranslated region (UTR) of firefly luciferase reporter mRNA, to be targeted by antisense vsiRNAs with EVs from NoV∆B2-immunized BALB/c suckling mice. A translation initiation codon-deleted GFP sequence of the same length was inserted into the same site as control.
Temporal profile of normalized intensity of total converted red fluorescence. Mean ± SEM is shown for n = 14 cells.
(C) Protein levels of IL-17A and IFN-γ from small intestine homogenates of CftrWT and CftrF508del/F508del (n= 10). Mean ± SD of triplicates of independent pooled samples. ***p<0.001 CftrWT vs CftrF508del/F508del (Student's t test).
C. T6SS secretion assay of various A. tumefaciens strains: wild type C58, ΔtssL, the tde double deletion mutant (Δtdei) containing pRL662 and pTrc200 empty vector (V) only or expression of pTdei1 (tde1-tdi1 expressed from pTrc200), pTde2* (catalytic site mutated tde2 expressed on pRL662), or pTdei1+ pTde2*.
(C) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7. (
G. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of CD4+ T cells isolated from OVA/OVA-immunized WT and CCR5-/- mice. Bottom, quantification (mean ± SEM) of gold particles in clusters of the indicated size (WT, gray bars; n = 5 cells, 14680 particles; CCR5-/-, red; n = 7 cells, 15374 particles). Insets show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.
(D) Microarray analysis of macrophage gene expression in IL-4C treated WT or Brd4 KO mice. Macrophages were isolated from peritoneal cells by the MACS system. Differentially expressed genes were identified with a cut-off line of log 2fold change >2 difference with p value of < 0.05. Red dots indicate differentially expressed genes (KO vs WT) and gray dots are those below the cut-off line.
C. Images (top panels) and corresponding contrast-enhanced MRI (middle and bottom panels) of the liver from representative Tie2-GFP mice or Tie2-IFNα mice that were intrahepatically injected with 5x103 CT26. White dashed lines identify macroscopic lesions. Red and green dashed lines identify hepatic or extrahepatic CRC metastases respectively from representative MRI z-stacks. Tumors detected by MRI analysis appeared as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences, respectively. Scale bars=5mm.