EMBO/sd-character-level-ner huggingface dataset:
AAV-BR1-mediated transduction of brain endothelial cells in vivo.Figure 5C57BL/6 mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.A Representative images from cerebellum, olfactory bulb, striatum, cerebral cortex, and the spinal cord, 14 days after vector injection. BR1-eGFP-transduced cells (green) were positive for the endothelial marker CD31 (red). Scale bars represent 250 µm. C57BL/6mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.B Higher magnification confocal microscopy images. The endothelial marker CD31 (red) colocalizes with vector-mediated eGFP expression (green). Scale bars represent 50 µm (upper panel) or 10 µm (lower panel). C57BL/6 mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.C CD13 staining of pericytes. The vector-mediated eGFP expression pattern (green) does not colocalize with CD13 (red). Scale bars represent 250 µm (upper panel) or 10 µm (lower panel). C57BL/6 mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.D Aquaporin 4 staining of astrocyte end-feet. The vector-mediated eGFP expression (green) does not colocalize with aquaporin 4 (red). Scale bars represent 100 µm (upper panel) or 10 µm (lower panel). C57BL/6 mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.E Expression of eGFP in primary brain endothelial cells prepared from C57BL/6 mice, 14 days after injection with AAV-BR1-eGFP. Scale bars represent 500 µm (upper panel) or 100 µm (lower panel).
Figure 3(A, B) UMAP visualization of whole embryo scRNA-seq for stages 8,10 and 12 colored by stage (A) and cell type annotations (B).
Figure 3A) Representative zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and REC8. Bellow: graph showing the percentages of Stag3 mutant axes with sites of LSAEs with and without flanking REC8 foci. 78 axes with LSAEs were analysed. Bars, 1 μm. B) Representative pachytene-like Rec8−/−, zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and RAD21L. Bars, 1 μm. C) Representative zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and RAD21. Bars, 1 μm. D) Schematic representation of univalents axes (black lines) with sites of LSAEs, and relative distribution of REC8 (green), RAD21L grey) and RAD21 (grey).
Figure 6A. Direct interactions between SidP and MavQ in the GST pull-down assay. GST beads coated with GST-SidP or GST were incubated with His6-MavQ. After washing the beads with GST binding buffer, the proteins resolved by SDS-PAGE were detected by Coomassie brilliant blue staining. B, SidP does not impair the kinase activity of MavQ in vitro. MavQ was preincubated with increasing amounts of SidP, and the kinase activity of MavQ was measured by the ADP-Glo assay (B) C. SidP does not impair the kinase activity of MavQ in vitro. MavQ was preincubated with increasing amounts of SidP, and the kinase activity of MavQ was measured by TLC assay (C). D. Co-expression of SidP with MavQ in HeLa cells does not influence the production of PtdIns3P caused by MavQ. The association of the fluorescence probe GFP-2xFYVEHrs with vesicle-like structures was used to indicate the distribution of PtdIns3P in cells. At least 100 cells were scored in each sample (n≥100) (left panel). Wort, wortmannin. Expression of MavQ and SidP in the samples were shown in the right panel. EV represents empty vector. E. Deletion of sidP does not impact the association of SidC with the LCV. BMDMs infected with the indicated L. pneumophila stains were stained and the association of SidC with bacterial phagosomes was similarly determined (n≥100) (left panel). Representative images of infected cells harboring bacterial phagosomes were also shown (right panel). Insets represent 3x magnification of regions defined by dash lines. Scale bar, 5 μm.
t-circles contain the C-rich telomere strand without subtelomeric Y' sequence.Figure 2A Senescence curve of a representative est2∆ clone and corresponding dot blot of the RCA product hybridized with the indicated subtelomeric or telomeric probes. t-circle amounts were evaluated based on signal intensity of the dots and were expressed as fold increase over the signal in the absence of Φ29 (lower graph). B Senescence curve of a representative est2∆ clone (upper panel). DNA samples were prepared at the indicated time points to analyze telomere length by Southern blot (middle panel) and t-circles production by RCA assay performed in the absence of dCTP and dATP.(lower panel). The graph shows signal intensities of the dots measured with the Fiji software (arbitrary units).
a, Samples from animals with salivary gland-specific knockdown of Atg12 (fkh–Gal4/w; UAS-Atg12IR/+), n = 21, and those with salivary gland-specific knockdown of both Atg12 and drpr-I (fkh–Gal4/w; UAS-Atg12IR/+; UAS-drpr-IIR/+), n = 19, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation.figf3a, Samples from animals with salivary gland-specific knockdown of Atg12 (fkh-Gal4/w; UAS-Atg12IR/+), n = 21, and those with salivary gland-specific knockdown of both Atg12 and drpr-I (fkh-Gal4/w; UAS-Atg12IR/+; UAS-drpr-IIR/+), n = 19, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. b, Quantification of data from a. c, GFP-LC3 was expressed in salivary glands of control animals (+/w; UAS-GFP-LC3/+; fkh-Gal4/+) and those with salivary gland-specific knockdown of drpr (w; UAS-drprIR/UAS-GFP-LC3; fkh-Gal4/+). Salivary glands were dissected 6 h and 14 h after puparium formation, imaged for GFP-LC3, and LC3 puncta were quantified using Zeiss Automeasure software. d, Quantification of data from c. Error bars represent s.e.m.; n ≥ 10; p  0.0000001. e, LAMP1-GFP was expressed in control animals (tub-LAMP-GFP/w; +/fkh-Gal4) and those with salivary gland-specific knockdown of drpr (tub-LAMP-GFP/w; UAS-drprIR/+; fkh-Gal4/+). Salivary glands were dissected 14 h after puparium formation, imaged for LAMP-GFP, and LAMP puncta were quantified using Zeiss Automeasure software. f, Quantification of data from e. Error bars represent s.e.m.; n ≥ 10; p  0.05. g, Samples from drpr mutant animals (+/w; +/UAS-Atg16A; drprDgr;5/drprDgr;5), n = 10, and those with salivary gland-specific expression of Atg16A (fkh-Gal4/w; UAS-Atg16A/+; drprDgr;5/drprDgr;5), n = 16, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. h, Quantification of data from g.
KRAS and TP53 alterations in MPM patients from France and Turkey.Figure 4Pleural fluid cell pellets and supernatants from 10 patients (called CRCINA #) with pleural effusion and pleural tumor samples from 17 patients (called TR#) with MPM were subjected to digital droplet polymerase chain reaction (ddPCR) for the detection of mutant (MUT) copies of KRAS codon 12/13 (KRASG12/13) and KRAS codon 61 (KRASQ61), as well as copies of TP53 and TERT. Diagnoses were lung adenocarcinoma (LUAD; n = 4) and MPM (n = 23). The assays were designed for detection of down to 1:20000 copies using EKVX (KRASWTTP53G610T), A549 (KRASG12STP53WT), NCI-H460 (KRASQ61HTP53WT), NCI-H3122 (KRASWTTP53E285V), and NCI-H3255 (KRASWTTP53G560-1A) human LUAD cells as controls. Shown are individual patient (KRAS plot) and individual sample (TP53 plot) allelic frequencies with color code and limits of normal TP53 allelic frequency as vertical dashed lines in the TP53 plot (A) Any number of KRAS-mutant droplets detected in any sample (KRAS plot in A) and any patient that failed to achieve normal TP53 ploidy by any sample (TP53 plot in A) was deemed altered. Pleural fluid cell pellets and supernatants from 10 patients (called CRCINA #) with pleural effusion and pleural tumor samples from 17 patients (called TR#) with MPM were subjected to digital droplet polymerase chain reaction (ddPCR) for the detection of mutant (MUT) copies of KRAS codon 12/13 (KRASG12/13) and KRAS codon 61 (KRASQ61), as well as copies of TP53 and TERT. Diagnoses were lung adenocarcinoma (LUAD; n = 4) and MPM (n = 23). The assays were designed for detection of down to 1:20000 copies using EKVX (KRASWTTP53G610T), A549 (KRASG12STP53WT), NCI-H460 (KRASQ61HTP53WT), NCI-H3122 (KRASWTTP53E285V), and NCI-H3255 (KRASWTTP53G560-1A) human LUAD cells as controls. results summary table (B). Any number of KRAS-mutant droplets detected in any sample (KRAS plot in A) and any patient that failed to achieve normal TP53 ploidy by any sample (TP53 plot in A) was deemed altered.
Figure 2(A) Examples of chromosome arms exhibiting features of telomeric semiconservative (Semi) replication, conservative (Consrv) replication of the entire telomere (E) and conservative replication of part of the telomere (P). Idealized chromosome diagrams (left) and actual microscopy images (right) are shown. D-FISH, denaturing FISH; ND-CO-FISH (non-denaturing CO-FISH).(B) Percentages of chromosome arms exhibiting conservative (Consrv) replication of the entire telomere (E), conservative replication of part of the telomere (P) and conservative replication of either the entire telomere of part of it (E+P). NE, U2OS cells expressing normal levels of cyclin E; OE, U2OS cells overexpressing cyclin E for four days. Bars represent means and standard errors of the mean from three independent experiments. Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres: P<0.025 for Consrv (P) and P<0.05 for Consrv (E+P), as calculated using the paired t test.
Figure 1Schematic representation of NUP58, CHMP1B, NUP85 and NUP153 genes (transcript IDs reported: NUP58 ENST00000381736.8, CHMP1B ENST00000526991.3, NUP85 ENST00000245544.9, NUP153 ENST00000537253.5). Exons shown as black boxes, introns as connecting black lines, and gRNA target sites are indicated with red triangles. Scale bars, 2kb. Diagram detailing experimental method for isolation of mutant clones. After transfection with eGFP Cas9 plasmid carrying dual gRNA, GFP+ HAP1 cells were individually sorted into 96-well plates. A total of 10 plates were prepared for each biological replicate (n=3). Relative cellular viability following Cas9-induced gene editing. Reported number of colonies was normalized based on viability of HAP1 cells transfected with an Empty Vector (EV) control (dots). Error bars indicate mean of SD from three biological replicates (number of colonies for EV rep1=242/rep2=238/rep3=201; CHMP1B rep1=134/rep2=151/rep3=128; NUP58 rep1=11/rep2=13/rep3=15; NUP85 rep1=0 /rep2=0/rep3=5; NUP153 rep1=61/rep2=55/rep3=60)(Ordinary one-way ANOVA ****P ≤ 0.00001; ns = non-significant). Graph indicates the percentage of viable clones with either undetectable, truncated or wild-type-like proteins as assessed by western blot analysis. Percentage of not analysed colonies is reported after CHMP1B targeting.
Fig.Figure 4A. Structural alignment of ubiquitin (magenta) from the MvcA-UBE2N-Ub complex and Ub (yellow) from CHBP-Ub (PDB code:4HCN), the carboxyl tails are indicated by a dashed box. B. Overall structure of MvcA(green)-ubiquitin(magenta) from the complex in comparison with Apo MvcA(marine). The conformational changes are indicated by two arrows, the black one indicates the shift of the tail domain and the red one indicates the rotation of the insertion domain. The potential clash areas between Apo MvcA and ubiquitin are indicated by a dashed circles. C. The five contacting interfaces between MvcA(green surface) and ubiquitin(magenta), each is indicated by a dashed circle and a letter from A-E. The image on the right is obtained by a 180° rotation of the left structure around the indicated axis. The interaction patch involving the C-terminal tail of ubiquitin is indicated by dashed circle. CTC: carboxyl terminus contacts. Detailed views of contact areas A (D) Key residues involved in interactions between MvcA and ubiquitin are labeled and shown as sticks. The hydrogen bonds are shown as dashed lines. Detailed views of contact B (E), contact C (F), contact D (G) and the carboxyl terminus contact (CTC) (H). Key residues involved in interactions between MvcA and ubiquitin are labeled and shown as sticks. The hydrogen bonds are shown as dashed lines. I-J. The roles of ubiquitin contact residues in the activity of MvcA. I. The indicated amounts of MvcA or its mutants were incubated with 2 μg UBE2N-Ub for 5 min in 25 μl reactions. The ratio of cleavage was analyzed by SDS-PAGE and then densitometry. Each bar represents the ratio of cleavage obtained from three independent experiments. Bars and error bars: means and standard error of the mean (SEM). J. The activity of the MvcA mutant harboring three mutations. 0.05 μg, 0.25 μg and 1.25 μg of MvcA or the mutant was incubated with 2 μg UBE2N-Ub in 25 μl reactions. After 30 min incubation, the products resolved by SDS-PAGE were detected by CBB staining. Note that the mutant did not detectable cleave UBE2N-Ub even in the reaction received the highest amount of protein.
Figure 6Binding sites of mabs (highlighted with ovals) were mapped by mutational analysis (A1 and A2) or by neutralization escape (A3 and B4). The residues affecting binding are represented as white patches on the surface of the sE trimer (PDB 1URZ) color-coded as domains. The a-d surface segments identified by the HDX-MS experiments are shown as grey patches. Blocking ELISA with mabs A1, A2, A3 and B4. Serial dilutions of the trimers were pre-incubated with a predetermined concentration of the respective mabs. The fraction of mab not blocked by the antigen was detected in ELISA with TBEV-coated plates (Materials and Methods). The bar charts show the results expressed relative to the reactivity with the sE401v trimer. Data are from at least three independent experiments.
a) Hematoxylin and eosin staining of the dentate gyrus (DG) and SVZ from Ctrl and FIP200GFAP cKO mice at P28.figf2(a) Hematoxylin and eosin staining of the dentate gyrus (DG) and SVZ from Ctrl and FIP200GFAP cKO mice at P28. Lines indicate the boundaries of the DG and SVZ. Arrows mark cells in the SVZ. (b-e) Immunofluorescence for GFAP, nestin or SOX2 and DAPI in the DG (b,c) from Ctrl and FIP200GFAP cKO mice at P28. The boxed areas in c-e are shown in more detail in insets (c). Dotted lines indicate boundaries of the granular zone (GZ) and SGZ (b). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (b,c), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. (b-e) Immunofluorescence for GFAP, nestin and DAPI in the SVZ (d,e) from Ctrl and FIP200GFAP cKO mice at P28. The boxed areas in c-e are shown in more detail in panels below (d,e). Dotted lines indicate boundaries of the SVZ (d,e). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (b,c), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. (b-e) Immunofluorescence for GFAP, nestin or SOX2 and DAPI in the SVZ (d,e) from Ctrl and FIP200GFAP cKO mice at P28. The boxed areas in c-e are shown in more detail in panels below (d,e). Dotted lines indicate boundaries of the SVZ (d,e). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (b,c), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. (f) Immunofluorescence for PSA-NCAM in the RMS (boxed areas), SVZ and DG of Ctrl and FIP200GFAP cKO mice at P28. Lines indicate the boundaries of the DG and SVZ. Arrows mark PSA-NCAM+ cells in the RMS, SVZ and DG. (g) Immunofluorescence for NeuN in the olfactory bulb (OB) and DG of Ctrl and FIP200GFAP cKO mice at P31. Lines indicate the layers of the OB and boundaries of the DG. Boxed areas in top panels are shown in more detail for staining of NeuN in insets. CC, corpus callosum; E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer.
Systemic treatment with recombinant IL-13 rescued the regenerative defects in CM-G4-KO mice.Figure 5(A) Depiction of the experimental time scale. (B) Quantification of left ventricular (LV) scar area of mice 7 days after cryoinjury and treated as indicated. The number within bars indicates the number of mice analyzed in that particular group. **p=0.0017 between Con and CM-G4-KO after PBS treatment and p=0.0078 between CM-G4-KO mice with PBS or IL-13 treatment. (C) Echocardiography analysis of left ventricular systolic function in the indicated mice 7 days after cryoinjury; *p=0.0218 (D) Quantification of Ki67 or pH3 positive cardiomyocytes in mice treated as described in (A); *p=0.0106 and **p=0.0052 between Con and CM-G4-KO after PBS treatment and *p=0.0439 or **p=0.0023 between CM-G4-KO mice with PBS or IL-13 treatment. (E) Quantification of capillary density as capillary/cardiomyocyte (CM) ratio in mice treated as described in (A). *p=0.0365, **p=0.0031. (F) Quantification of α-SMA positive small conductance vessel (~20-50µM in diameter) in myocardial sections of the mice treated as indicated. (G) Representative immunoblots for STAT6 and GAPDH from neonatal mouse hearts (KO = CM-G4-KO) treated as indicated. The quantification of the blots is shown on the right. *p=0.0114.
Figure 4A. Drp1 oligomerization by chemical crosslinking in wild-type CNs with or without rIFN-β for 2 h. Drp1 oligomerization by chemical crosslinking in wild-type CNs with or without rIFN-β for 2 h. B. Quantification of A. C. Immunoblot of INF2 and quantification from cell fractionation of Ifnb+/+ and Ifnb-/- CNs with or without rIFN-β for 2 h. D. EM of Ifnb+/+ and Ifnb-/- thalami from 12-month-old mice. Scale bars equal 5 microns. Black arrows indicate mitochondria at a distance < 200 nm, and white arrows indicate distance >200 nm. E. Volume rendering from 3D TEM images of Ifnb+/+ and Ifnb-/- thalami. Mitochondria are blue, the ER is yellow, and nuclei are grey. Mitochondria undergoing fission are cyan and indicated with a white arrow. 2D pictures are shown in Fig 2. F. ER/mitochondria closeness, evaluated as the distance from a mitochondrion to the nearest ER structure, extracted from EM images. G. Contact zone length, quantified as the percentage of the perimeter of a mitochondrion in close contact (<20 nm) with ER, extracted from EM images. H. Projections of 3D images from immunostaining of Drp1 (blue), Hsp60 (mitochondria, green), INF2 (yellow), and calnexin (ER, magenta) in Ifnb-/- MEFs with or without rIFN-β treatment for 6 h. Scale bar equal 2 microns. Arrows indicate fission foci. I. Quantifications of triple-positive MT-DR+Drp1+Cnx+ in MEFs. Images are shown in Fig EV3.
Transient expression of SipB in BMDPM from caspase-1−/−mice results in cell death.Figure 2. Transient expression of SipB in BMDPM from caspase-1−/−mice results in cell death. Plasmids expressing either SipB, SipC, or SipD fused to YFP were introduced into BMDPM from caspase-1−/− mice as indicated in the Materials and methods section. Macrophages were then fixed, stained with DAPI (to visualize chromatin), and observed under a fluorescence microscope. Cells expressing SipC or SipD do not show any signs of cytotoxicity. In contrast, cells expressing SipB show clear signs of cytotoxicity and chromatin condensation. Arrows indicate the nucleus of transfected cells.
Liquid-liquid phase separation of p62 is affected by MOAP-1.Figure 4A Larger RFP-p62 bodies in the MOAP-1 deficient cells. WT and MOAP-1 KO LO2 cells were transfected with expression vector encoding RFP-tagged p62. 16 hours later, cells were subjected to live imaging analysis. Z-stacks of the confocal images were flattened as maximal intensity projections. Scale bar: 5 µm.C Western analysis of lysates of the WT and MOAP-1 KO LO2 cells transfected with expression vector encoding RFP-tagged p62 as in (A). D, E RFP-p62 bodies exhibited fusion (D) and fission (E) characteristics of liquid droplets. WT and MOAP-1 KO LO2 cells were transfected with expression vector encoding RFP-tagged p62 and 14 hours later, cells were analyzed by live-imaging to visualize the dynamics of RFP-p62 bodies. Scale bar: 2 µm F Loss of MOAP-1 leads to slower exchange of p62 in the RFP-p62 bodies. WT and MOAP-1 KO LO2 cells transfected with expression vector encoding RFP-tagged p62. 14 hours later, cells were subjected to the fluorescence recovery after photobleaching (FRAP) assay. Scale bar: 2 µm.H Overexpression of MOAP-1 but not GFP leads to increased exchange of p62 in the RFP-p62 bodies. HepG2 cells were transfected with expression vector encoding RFP-tagged p62 and GFP-tagged MOAP-1 or GFP only control and 14 hours later, cells were subjected to FRAP assay. Scale bar: 2 µm.J Re-introduction of MOAP-1 disrupts the spherical structure of p62 bodies in the MOAP-1 deficient LO2 cells. MOAP-1 KO LO2 cells were transfected with expression vector encoding Myc-MOAP-1 and harvested at 14- or 24-hours post-transfection for IF analysis using anti-p62 (in red) and anti-Myc (in green) antibodies. Insets were enlarged in the lower panels. Scale bar: 2 µm.
Fig.Figure 2(A) In vivo characterization of mitochondrial targeting capacity of different Mrp17 truncations fused to GFP visualized by fluorescent microscopy with MitoTracker Orange straining. Scale bar for all micrographs is 10 µm. (B) Characterization of Mrp17 translocation signal using an in vitro import assay: shown are autoradiographs of full-length Mrp17 or its truncations fused to DHFRmut, translated in vitro with radiolabeled amino-acids, incubated with isolated yeast mitochondria for 2, 5, or 10 min, treated with proteinase K (PK) to remove nonimported proteins and visualized by 16% SDS-PAGE/autoradiography. As a negative control, mitochondria were treated with valinomycin, antimycin and oligomycin (VAO) that eliminate membrane potential. For comparison, 20% of the protein used per import reaction was loaded on the first lane.