EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 5(A) Network analysis of 66 potential CLPP substrates using only high confidence interactions (> 0.7). Known ClpXP substrates are shown in boxes with a green frame. Respiratory chain proteins and ribonucleoproteins are indicated in dark grey and orange, respectively. (B) Western blot analysis and subsequent quantification of ERAL1 and EFG1 levels from Clpp knockout (Clpp-/-) heartmitochondria relative to wild type (Clpp+/+). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=4). The arrowhead indicates positions of EFG1. (C) Expression levels of potential CLPP substrates from Clpp knockout (Clpp-/-) heartmitochondria relative to wild type (set to 1). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=4). (D) Cycloheximide (CHX) chase assay to determine the stability of ERAL1 and EFG1. Proteins are visualized by Western blot with respective antibodies. Proteins are isolated after 2, 4, 6, 8,10 and 12 hours after beginning of chase (0). The arrowhead indicates positions of ERAL1. (E) Western blot analysis and subsequent quantification of protein levels in wild type (C1, C2) and Clpp knockout (KO1, KO2) MEFs upon Clpx siRNA (Clpx) relative to untreated (Ctrl) wild type. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=6). The arrowhead indicates positions of either CLPX or ERAL1, and stars indicate position of cross-reacting bands.
Figure 3Representative images of CXCR4 overexpression in subcutaneous tumor tissue, showing similar CXCR4 levels among compared groups (N=5/group; Buffer, T22-GFP-H6-FdU, T22-GFP-H6 and free oligo-FdU) before treatment (upper panels). Representative images of DNA double-strand break induction and caspase 3 activation (measured with anti-ɣH2AX or anti-cleaved caspase 3 by IHC) 5h post-administration (middle panels). Apoptotic induction (Hoescht staining, 24h post-administration, lower panels). Note the higher number of cells positive for DSBs, Caspase 3 activation and apoptosis induction in the T22-GFP-H6-FdU as compared to free oligo-FdU. Black or white arrows indicate dead cells. Quantitation of the number of cells containing DSBs or active Caspase-3 in IHC-stained tumor sections 5h post-treatment and the number of condensated or disaggregated nuclei (by Hoescht staining) 24h post-treatment in tumor sections of 10 high-powder fields (400x magnification) using the Cell D software. (N=50; 10 tumor fields/mice; 5 mice/group). Data expressed as mean±s.e.m.
Cancer-derived EVs suppress AA-induced responses of an mTORC1 activity reporter.Figure 2A. Schematic of the FRET reporter for mTORC1 activity (TORCAR). B. TORCAR FRET reporter assay in WI-38 or NIH3T3 cells after 2-h starvation and EAA add-back at time 0 (n=3 biological replicates for each group). Data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA).
Selective inhibition of telomeric DDR decreases ACE2 expression in cultured cells and in vivo.Figure 5A Immunofluorescence showing gammaH2AX foci in Trf2F/F MEFs at the indicated time points following 4OHT treatment and consequent TRF2 knockout and treated with DMSO or ATMi. Scale bar, 25 µm. B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments). C Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce telomere uncapping) or vehicle and injected with the indicated ASOs or PBS as control. Scale bar, 10 µm. D RT-qPCR detection of Ace2 mRNA levels in livers of mice treated as in C (n = 5-8 mice per group). E Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 200 µm. F Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive cells in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.
Figure 4(a) Effect of compound WX-340 on binding of recombinant murine uPA to PAI-1 protein as assessed by ELISA, quantitative data are shown (mean±SEM for n=3 experiments per group; #p<0.05 vs. drug vehicle; One-way ANOVA). (b) Intravascular rolling and firm adherence as well as transmigration of neutrophils and classical monocytes (cMOs) to the inflamed perivascular tissue as assessed 6 h after intrascrotal injection of TNF in postcapillary venules of the cremaster muscle of WT mice, quantitative data are shown (mean±SEM for n=6 mice per group; #p<0.05 vs. saline; *p<0.05 vs. drug vehicle; One-way ANOVA). (c) Expression of uPA or PAI-1 (green) in tumors as assessed in an orthotopic model of 4T1 breast cancer in WT mice by confocal microscopy, PECAM-1/CD31+ postcapillary venules (blue) and Ly-6G+ neutrophils (red) are depicted (scale bar: 100 µm). (d) Relative development rates and tumor weight in animals treated with WX-340 a priori or therapeutically after 1 week after tumor cell injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; One-way ANOVA). (e) Intravascular rolling and firm adherence of neutrophils as assessed in the tumor microvasculature 14 days after intra-dermal injection of 4T1 cells in the right ear of WT mice treated with WX-340 or vehicle by multi-channel in vivo microscopy, quantitative data are shown (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; t test). (f) Numbers of neutrophils and 4T1 tumor cells in lungs and brain of WT mice receiving WX-340 or vehicle therapeutically after 1 week after tumor cell injection on a daily basis as assessed 14 days after intravenous injection of 4T1 tumor cells by multi-channel flow cytometry, quantitative data are shown (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; t test).
Eis modulates ROS release and proinflammatory cytokine production through its N-acetyltransferase domain.Figure 7(A) Intracellular superoxide production was analyzed by flow cytometric analysis. BMDMs were infected with Mtb-Δeis (MOI = 10) in the presence or absence of recombinant Eis protein (rEis; 5, 10, 20 µg/mL) or 30 kDa Mtb antigen (30 k; 5, 10, 20 µg/mL). Upper, representative flow cytometric analysis; lower, quantitation of superoxide generation. (B) THP-1 cells were transfected with mock, Eis-WT, or Eis-ΔAT constructs, and infected with Mtb-Δeis for 30 min. Cells were stained with DHE (for superoxide) or DCFH-DA (for H2O2) and subjected to flow cytometric analysis. Inset, transfection efficiency. (C and D) Experimental conditions were identical to those outlined in panels A and B, respectively. Supernatants were collected 18 h after infection and assessed by ELISA for levels of TNF-α and IL-6. Data are presented as the mean±SD of five experiments. (E and F) THP-1 cells transfected with mock, Eis-WT, or Eis-ΔAT constructs were pretreated with SP600125 (SP; 20 µM) or SB203580 (SB; 5 µM) for 45 min before infection with Mtb-Δeis for 30 min (E) or 18 h (F). E, Intracellular superoxide production was analyzed by flow cytometric analysis. F, ELISA analysis for TNF-α and IL-6 levels. Data are presented as the mean±SD of three experiments. **p<0.01, ***p<0.001, vs. SC (A, C, E, and F); mock control (B and D). UI, uninfected; SC, solvent control (0.1% DMSO).
TRIM21 is involved in inflammasome activation rather than enhancing pro-IL-1β expression.Figure 5HMDM were stimulated with AdV (50 000 pp/cell) and antibody (h9C12; 20 ug/ml, IVIg: 20 mg/ml) or 10 μg/ml pI:C for 3h and gene expression was measured by qPCR (n = 4 mean ± s.e.m). HMDM were stimulated as in (A), or with 10 ng/ml LPS for 6 h and pro-IL-1β levels in the cytosol measured by western blot. Blot is representative of three independent donors. HMDM were stimulated as in (A), or with 10 ng/ml LPS for 3 h and NLRP3 mRNA expression measured by qPCR. Data shows average ± s.d. of 2 independent donors HMDM were stimulated as in (B) and NLRP3 levels in the cytosol measured by western blot. Blot is representative of two independent donors. TLR9 mRNA levels from PBMCs, CD19+ve B cells, CD14+ve monocytes or HMDM derived from CD14+ve monocytes were assessed by qPCR and copy number was determined relative to actin copy number (n = 5 mean ± s.e.m). THP-1s expressing ASC-GFP were stimulated for 6 h with virus and antibody or 200 ng/well HT-DNA or 10 μM Nigericin, in the presence of the pan-caspase inhibitor zVAD-fmk. A representative image (scale bar 100 μm) and quantification of number of cells with ASC specks from three independent experiments (mean ± s.e.m, *P-value ≤ 0.05, paired, two-tailed t-test) is shown.
Figure 3(A, B, C, D) Cells were treated with MDMX siRNA for 48 hrs and analyzed for β-catenin pS45 level by western blot. (E) H1299 and 293T cells were treated with indicated siRNA for 48 hrs. The activity of β-catenin S45 kinase in cell extracts was determined using an ELISA assay. The results are average of 3 experiments (mean +/- SD). *p<0.05 (Student's t test).
Identification of anti-PrP Fab71 analogous antibodies in human antibody repertoires.Figure 4(A) Fab71-similar HCDR3 sequences in three different NGS datasets (DW: dataset for VH:VL; BB and DK datasets for VH) of human antibody repertoires from healthy donors. Pie chart and stacked bar plot (middle) indicate the occurrence of the identified Fab71 similar HCDR3s: 555 out of 629 identified sequences were only found in one donor, while 74 sequences were shared between donors and occurred in 2 up to 6 donors. Stacked bar plots (right) report the number of sequences differing to Fab71 HCDR3 by 3, 2 or 1 residues. In total, 13 sequences differed by only one residue. Among them, 4 sequences were shared between different donors. (B) Sequence alignment of Fab71 VH3-30 with HCDR3 regions from the different databases that differ from Fab71 by ≤ 3 residues (upper panel). Amino acids differing from Fab71 are highlighted by orange boxes. Lower panel: sequence alignment of Fab71 light chain VK3-15 with DK_VL2-14 that is naturally paired with DK_VH3-07. (C) Left: Heat map showing the binding specificities (Z-scores) of selected Fab71 human analogous antibodies to human recPrP23‑230 compared to the negative controls (BSA and Neu). Right: Heat map representing the reactivity (-logEC50) obtained from dose-dependent ELISA binding curves of the analogous antibodies to human recPrP23‑230. Red: low reactivity; blue: high reactivity.
Figure 1 Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (A) Percentage body weight loss Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (B) Relative amount of PR8 viral NP mRNA, by qPCR Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (C) Histology of lungs by H&E staining with pathology scores. Scale bars = 200 μm Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (D) Absolute number of live CD45+ cells in whole lungs and BAL, by flow cytometry Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M Statistical significanc was determined with unpaired student's T-test  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (E) Level of LDH in BAL, by LDH cytotoxicity assay Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (F) Expression of PDPN in lungs, by western blot Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (G) Total protein in BAL, by Bradford assay. (H) Concentration of albumin in BAL, by ELISA Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M. Statistical significanc was determined with unpaired student's T-test
a) Fluorescence microscopy of DHE→Eth conversion and annexin V (green)/PI (red) co-staining of wild-type cells at day 18 of the chronological ageing experiment shown in Fig.figf3(a) Fluorescence microscopy of DHE→Eth conversion and annexin V (green)/PI (red) co-staining of wild-type cells at day 18 of the chronological ageing experiment shown in Fig. 1b. Scale bars, 10 μm. (b, c) Quantification of DHE→Eth conversion (b) and annexin V/PI co-staining (c) using FACS analysis performed at indicated time-points of the chronological ageing experiment shown in Fig. 1b. Data represent means ± s.e.m. (n = 3; *P 0.001). (d) Electron microscopy of young log-phase cells (day 0) and of 20-day-old wild-type cells aged without (control) or with spermidine (4 mM). Representative cells are shown (for an overview see Supplementary Information, Fig. S3c). (e) Fluorescence microscopy of chronologically aged wild-type cells (day 3 and 14) expressing an EGFP-tagged version of the yeast HMGB1 homologue (Nhp6A-EGFP) with or without (control) addition of 4 mM spermidine. Scale bars, 5 μm.
Figure 6Phylogenetic tree of COP1 homologs from 100 species showing the presence of the pseudosubstrate latch (PSL) in COP1 and the presence of Tribbles homologs in the indicated species.
Figure 3(A) Zoomed-in view of site IV. Residues of hCAP-G and hCAP-H are shown in orange and green, respectively. The dashed red lines indicate hydrogen bonds. (B) 3Q, 5Q, 3A, 5A, 2A and Δ506-515 mutants of hCAP-H. Motif IV (residues 461-503) contains amino-acid residues highly conserved among eukaryotic species (X, Xenopus laevis; Dr, Danio rerio; Cm, Cyanidioschyzon merolae; Sp, Schyzosaccharomyces pombe; Sc, Saccharomyces cerevisiae; Ec, Encephalitozoon cuniculi). To produce the IV-3Q, 5Q, 3A, 5A and 2A mutants, the conserved aromatic amino-acid residues (F463, F469, F473, F501 and Y503; labeled in dark blue) were substituted with glutamine (Q) or alanine (A) residues. The secondary structural element of hCAP-H is drawn below the sequence alignments. (C) Interaction analysis between hCAP-G and hCAP-H. Bacterial cell lysates co-expressing hCAP-G (residues 1-478, 554-900) and hCAP-H (residues 394-515), either wild type (WT; lanes 2, 10 and 13), 3Q (F463Q, F469Q and F473Q; lane 3), 5Q (F463Q, F469Q, F473Q, F501Q and Y503Q; lane 4), 3A (F463A, F469A and F473A; lane 5), 5A (F463A, F469A, F473A, F501A and Y503A; lane 6) or 2A (F501A and Y503A; lane 11), or a C-terminal deletion mutant (506-514 residues were deleted from 394-515; lane 14) were applied to Ni-NTA agarose resin, and the bound fraction was analyzed by SDS-PAGE. Alternatively, a cell lysate co-expressing mutant hCAP-G (D647K) and wild-type hCAP-H was examined (lane 7). The uninduced cell lysate was also used as a negative control (lane 9).
Modulation of miR-29 defines murine VSMC proteome and collagen targets .Figure 4Proteome studies analyze the frequency distribution of protein changes after the transfection of primary murine aortic VSMCs with pre-miR-29b, anti-miR-29b, pre-miR-30c, or anti-miR-30c. A log2 fold change of −1 or 1 was used as cutoff. Log2 (fold change) is calculated using the normalized spectral counts and averaged for the 3 biological replicates. (A) The log (fold change) for differentially secreted proteins following overexpression or inhibition of pre-miR-29b was analyzed, and significantly changed miR-29 confirmed target transcripts are shown. FDR indicates false discovery rate. The complete list of significant proteins is in (Appendix Table S1A-B). (B) Thirty microliters of conditioned medium from VSMCs transfected with pre-miR-29b and anti-miR-29b were separated by SDS-PAGE before validation of COL3A1 by immunoblotting. (C) Densitometry analysis of COL3A1 signal from blot in (B) (pre-miR, left *p<0.001, anti-miR, right *p=0.002) . All data in (C) represent the mean + SEM, *p<0.05 versus other groups in anti- and pre- groups. Level of significance was determined using one-way ANOVA with Bonferroni's post test. (D) Working model of LNA-miR-29 effects on plaque composition as detailed in the data of this paper. Administration of LNA-miR-29 blocks lesion growth and increases intra-plaque collagen expression and fibrous caps, two indices characteristic of more stable plaques. This panel was produced using Servier Medical Art (www.servier.com).
Arsenic induces DNMT3A degradation via the ubiquitin-proteasome pathway.Figure 3A Immunoblotting of DNMT3A protein levels in MEFs (upper panel) and CCRF-CEM cells (lower panel) treated with ATO in the indicated conditions. B MEFs (upper panel) and CCRF-CEM cells (lower panel) were pretreated with 20 μM DMSO, 1 μM MG132, 0.02 μM Concanamycin A (Conc A), 20 μM Calpeptin (Calp), or 20 μM Z-VAD-FMK (Z-VAD) for 2 hours, followed by 16-hour ATO co-treatment at 1 μg/mL for MEFs or 0.3 μg/mL for CCRF-CEM cells. Immunoblotting of DNMT3A in whole cell lysate is shown. C HCT116 cells were pretreated with 3 μM MG132 for 2 hours, followed by ATO co-treatment for 16 hours at the indicated concentrations. Immunoblotting of DNMT3A in whole cell lysate is shown. D In vivo ubiquitination assay. 293T cells were transiently transfected with HA-ubiquitin and Myc-DNMT3A. After 24 hours, cells were treated with or without MG132 for 2 hours at the indicated concentrations, followed by ATO co-treatment for 6 hours at the indicated concentrations. Myc-DNMT3A was immunoprecipitated (IP) with anti-Myc antibody, followed by immunoblotting (IB) as indicated. E Immunoblotting of the transfected wild-type DNMT3A, C520A/C524A and C541A mutants in 293T cells upon 1 μg/mL ATO treatment for the indicated durations. F Measurement of DNMT3A-ADD thermodynamic stability upon ATO or AsPh3 treatment. Purified DNMT3A-ADD was mixed with ATO or AsPh3 at the indicated ratios in 20 mM HEPES (pH 7.5) for 1 hour. Chemical structure of AsPh3 is shown. Melting curves were measured by DSF (upper panel), and the calculated ΔTm values are shown (lower panel). G Immunoblotting of DNMT3A in MEFs (upper panel) and CCRF-CEM cells (lower panel) treated with ATO or AsPh3 in the indicated conditions. H, I Immunoblotting of endogenous DNMT3A, DNMT3B and DNMT1 protein levels in CCRF-CEM (H) and THP-1 (I) cells treated with DAC or ATO in the indicated conditions. DAC: +, 0.2 μM. ATO: +, 0.06 μg/mL; ++, 0.2 μg/mL; +++, 0.6 μg/mL. Red asterisk indicates the specific band.
a,b) Autophagic clearance of Agm (a) or Agg (b) formed by FL WT and mutant K385R or K394R GFP-Sph1 in SY5Y cells, in response to serum removal (a) or upon blockage of basal autophagy with 3-MA (b).f8(a,b) Autophagic clearance of Agm (a) or Agg (b) formed by FL WT and mutant K385R or K394R GFP-Sph1 in SY5Y cells, in response to serum removal (a) or upon blockage of basal autophagy with 3-MA (b). (c,d) Left: Agm (c) or Agg (d) of FL GFP-Sph1 (green) immunostained for endogenous K63-linked ubiquitin (red) in SY5Y cells. Arrows in (c) indicate colocalization. Right: percentage of Agm (c) or Agg (d) positive for K63-linked ubiquitin. (e) Formation of WT and mutant K385R FL Sph1 (left) and ANK1-p38 (right) Agm in response to proteasome inhibition. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition (a,b) or WT FL Sph1 or ANK1-p38 (c-e). (f) Schematic model of the contribution of the ANK1 region and K63 linkage of this region to removal of Agg and Agms through different types of autophagy.
a) Schematic of WDR45, which comprises 12 exons (rectangles).figf1(b) RT-PCR analysis using the blue primer set (left) and green primer set (right) from a. Whereas control cDNA samples showed a single product corresponding to the wild-type allele (WT), an apparently longer product was observed in subjects 1, 2 and 5, indicating that only the transcripts from the mutant allele were expressed. In subject 3, both wild-type and mutant alleles were expressed. Template without reverse transcriptase was used as a negative control, RT(-).
PPAR/ activates the expression of UV-induced epidermal miR-21-3p.Figure 1(A) RT-qPCR quantification of relative miR-21-3p and miR-21-5p levels in mouse brain, heart, kidney and epidermis. N=4 animals per group, one representative experiment is shown out of three independent replicates. (B) Fluorescent miR-21-3p in situ hybridization (pink) in dorsal skin of acutely irradiated (Ac-UV) and non-irradiated (no UV) Ppard +/+ and -/- mice. E: epidermis; D: dermis; HF: hair follicle. Magnification bar: 100m (C) RT-qPCR quantification of relative pri-miR-21 and miR-21-3p levels in the epidermis (left and middle), and of relative miR-21-3p in the dermis (right) of acutely irradiated (Ac-UV; +) and non-irradiated (-) Ppard +/+ and -/- mice. Pri-miR-21: Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.022; miR-21-3p: Ppard +/+ no UV vs Ppard +/+ Ac-UV P = 0.017, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.008. N=3-4 animals per group, one representative experiment is shown out of three independent replicates. (D) RT-qPCR quantification of relative miR-21-3p levels in total skin of chronically irradiated (Chr-UV; +) and non-irradiated (-) Ppard +/+ and -/- mice. miR-21-3p: Ppard +/+ no UV vs Ppard +/+ Chr-UV P = 0.008, Ppard +/+ Chr-UV vs Ppard -/- Chr-UV P = 0.002, ns: non significant. N=4 animals per groups, one representative experiment is shown out of two independent replicates. (E) RT-qPCR quantification of relative miR-21-3p level in the epidermis of Ppard +/+ and -/-mice, acutely irradiated (Ac-UV; +) or non-irradiated (-), treated with the PPAR/ antagonist GSK0660 (+) or vehicle (-), as indicated. miR-21-3p: Ppard +/+ no UV vs Ppard +/+ Ac-UV P = 0.005, Ppard +/+ Ac-UV vs Ppard +/+ Ac-UV/GSK0660 P = 0.04, N=5 (Ppard +/+) to 3 (Ppard -/-) animals per group. One representative experiment is shown out of two independent replicates (F) RT-qPCR quantification of relative miR-21-3p levels in HaCat cells treated with the PPAR/ agonists GW501516, GW0742 (+) or vehicle (-) as indicated. miR-21-3p: Veh vs GW501516 P = 4E-04, Veh vs GW0742 P = 2E-04. N=2-3 biological replicates, one representative experiment is shown out of two independent replicates.
Figure 7(a) Cohesin generates and stabilizes DNA loops to organize interphase chromatin into topological domains. (b) Random DNA strand passage activity of topo II can either remove or produce DNA entanglements within and across such topological domains. Juxtapositions of DNA segments within a loop can lead to the formation of knots (i), whereas juxtapositions of DNA segments belonging to nearby loops or adjacent domains can lead to the formation of intra- (ii) or inter-molecular (iii) DNA interlinks. (c) To minimize the occurrence of these entanglements, condensin might use its DNA loop extrusion activity to constrict intra- and inter-molecular interlinks and so bias the DNA strand passage activity of topo II to remove them. This condensin function may operate during interphase to facilitate chromatin transactions and during cell division to enforce the removal of sister chromatid interlinks.