EMBO/sd-character-level-ner huggingface dataset:
OCRL localizes to the proximal end of centriolesA Representative images of RPE-1 cells co-stained with anti-OCRL (red) and anti-γ-tubulin (green) antibodies.Figure 4A Representative images of RPE-1 cells co-stained with anti-OCRL (red) and anti-γ-tubulin (green) antibodies. Insets show the enlarged centrosomal region. Scale bar, 10 μm. B Representative images of the co-localization of GFP-OCRL-WT (green) with centrosome marker γ-tubulin (red) in RPE-1 cells. Insets show the enlarged centrosomal region. Scale bar, 10 μm. C The purified centrosome fraction (50%) isolated from RPE-1 cells and the cell lysate (5%) were subjected to immunoblot analysis with anti-OCRL antibody. The centrosomal protein γ-tubulin was probed as control. D Representative images of RPE-1 cells co-stained with anti-OCRL (red) and anti-γ-tubulin (green) antibodies during mitosis. Insets show the enlarged centrosomal region. Scale bar, 2 μm. E Super-resolution images of centrosome stained with anti-CEP164 (red, labeling distal appendages of mother centriole) and anti-OCRL (blue) were acquired using Centrin2-GFP-RPE-1 cells by DeltaVision OMX V4 BLAZE system. Scale bar, 0.5 μm. F Super-resolution images of centrosome stained with anti-CNAP1 (red, labeling proximal ends of centriole) and anti-OCRL (blue) were acquired using Centrin2-GFP-RPE-1 cells by DeltaVision OMX V4 BLAZE system. Scale bar, 0.5 μm. G G Model depicting the localization of the OCRL on mother and daughter centrioles. H Representative images of the co-localization of OCRL-WT, GFP-OCRL-△RhoGAP and GFP-OCRL-△ASH+RhoGAP with γ-tubulin. Insets, enlargements of the outlined areas. Scale bars, 10 μm. I Quantitative analysis of cells with and without centrosomal OCRL (centrosomal GFP signal) in the experiments shown in A. n = 3 per group in which 10 cells were counted per condition per experiment. Means ±SEM, ****P < 0.0001, n.s., not significant, calculated by two-way ANOVA, multiple comparisons.
Figure 8A Seedling Cl- content determined by ion chromatography 2 h after treatment with 50mM NaCl. *p<0.05, Student's t-test; biological replicates: n=4. B Cl- content determined by ion chromatography of seedlings supplemented with 5mM KNO3 or NH4NO3. *p<0.05, Student's t-test; biological replicates: n=4. C On the left, relative Cl- content determined by XRF of NO3- starved seedlings after KNO3 supplementation. *p<0.05, Student's t-test; biological replicates: n=3-5. On the right, a representative XRF image of Cl- signal across WT and mutant roots at the 0h timepoint. Five roots per genotype are shown. The outlines indicate the measurement areas. The color legend indicates relative Cl- concentration. D Cl- and NO3- content determined by ion chromatography of transgenic roots of composite plants overexpressing MtNPF6.5 compared with control roots transformed with an empty vector (EV). The plants were grown on FP (NO3- starved) and then supplemented with 5mM KNO3 and sampled a 0 and 48h. *p<0.05, Student's t-test; biological replicates:n=3.
| Proteogenomics exploration for protein-level detection of isoforms, single amino acid variants and alternative translation sites.Figure 4Searching the tonsil proteomic data (trypsin alone or all enzymes) against a tissue-specific sequence database constructed from RNA-Seq data drastically reduces the number of individual protein sequences in protein groups compared to searches against Ensembl, allowing for the more efficient detection of protein isoforms. Number of single amino acid variants detected by whole exome sequencing and DNA, by RNA-Seq at the mRNA and by mass spectrometry at the protein level as well as confirmed candidates by validation using synthetic peptide spectra comparisons. It is apparent that only a very small fraction of all variants detected at the DNA or RNA level can be detected at the proteome level using current technology. Analysis of which proteomic workflow contributed to the detection and confirmation of single amino acid variants. Results of the detection of non-canonical coding regions using proteomics data (left panel) and different alternative start codons identified by acetylated N-terminal peptides (right panel). The majority of cases are N-terminal extensions of annotated genes. All but one of the detected alternative translation start sites correspond to point mutations of the first base of the classical AUG codon. Validation of a novel translation start site for the protein PLS3. The upper panel shows the novel translation site position within the amino acid sequence context and the lower panel shows a mirror plot of the tandem mass spectra of the endogenous N-terminally acetylated peptide (peaks pointing upwards) and the corresponding synthetic peptide spectrum (peaks pointing downwards). Y-type sequence ions are colored in red, b-type ions in blue and the intact peptide as well as neutral losses thereof are marked in yellow.
Figure 5Jurkat cells (a) were infected with EBV-WT or EBV-ΔBRLF1 virus (MOI=500) for 36 h. Then, the cells were collected, and the total RNAs were extracted, reverse-transcribed and analyzed by real-time PCR. Color gradient depicts the mean mRNA expression normalized to the housekeeping gene GAPDH and compared to the mock group. NK-92MI cells (b) were infected with EBV-WT or EBV-ΔBRLF1 virus (MOI=500) for 36 h. Then, the cells were collected, and the total RNAs were extracted, reverse-transcribed and analyzed by real-time PCR. Color gradient depicts the mean mRNA expression normalized to the housekeeping gene GAPDH and compared to the mock group. Jurkat and NK-92MI cells were cultured with conditional medium from EBV-WT or EBV-ΔBRLF1-infected Ramos cells (paracrine), or Jurkat and NK-92MI cells were directly infected with EBV-WT or EBV-ΔBRLF1 viruses (autocrine). (c) The cell pellets were collected and the whole cell extracts were analyzed by western blotting to detect TAK1, IKKα/β and IκB phosphorylation. The cell killing activity of Jurkat cells (d) were measured by the detection of the LDH release from the K562 target cells. The cell killing activity of NK-92MI cells (e) were measured by the detection of the LDH release from the K562 target cells. Anti-IL-1β and/or anti-IL-18 neutralizing antibodies (NA), TAT-Flag or TAT-N572 peptide were added into EBV-WT- or EBV-ΔBRLF1-infected Jurkat cells or NK-92MI cells for 24 h. (f) TAK1, IKKα and IκB phosphorylation were detected as indicated. cell killing activity of Jurkat cells (g) were measured. cell killing activity of NK-92MI cells (h) were measured.
TNF promotes fracture healing in osteoporotic bone.Figure 5rhTNF treatment augmented early phase fracture healing in osteoporotic mice. Treatment with 1 ng rhTNF at the fracture site on days 0 and 1 led to increased % callus mineralization at day 14 but no difference at day 28 indicating accelerated early healing but the same final result at day 28. Data are presented as mean ± SEM. At day 14, PBS control versus TNF, **P = 0.0099; at day 28, PBS control versus TNF, nsP = 0.37, by unpaired two-sided t-test. Representative micro-CT images at day 14 are shown. rhTNF treatment led to mature callus bridging across the fracture which was absent in the PBS-treated control group. Scale bar, 2 mm.
Molecular composition of reconstituted K.Figure 1A) Representative size-exclusion chromatography (SEC) chromatogram with absorbance measured at nanometers (nm) 260 or 280 (for this chromatogram and the following ones, absorbances are in units of 1000-1 (mAU)) of reconstituted K. lactis COMA-Nkp1-Nkp2, and image of Coomassie Blue-stained SDS-PAGE gel with fractions from principal SEC peaks. Molecular masses of protein standards (Mm. S.) are in kiloDalton (kDa); L: sample loaded on column. †: Escherichia Coli DnaK; ╪: proteolysed Nkp1-Nkp2; Nkp1-Nkp2 is less prone to spontaneous proteolysis when associated with COMA. B,C) Nanoflow mass-spectra of COMA-Nkp1-Nkp2 (B) acquired with different injected concentrations, showing an increase in COMA-Nkp1-Nkp2 dimers or COMA dimers with increasing injectant concentration. B,C) Nanoflow mass-spectra of COMA (C), acquired with different injected concentrations, showing an increase in COMA-Nkp1-Nkp2 dimers or COMA dimers with increasing injectant concentration. D) Graph with measured differential refractive index (relative scale) and molar mass calculated with multi-angle light scattering (MALS) data of COMA eluting from SEC (SEC-MALS). We injected COMA at a concentration of ~ 43 μM (COMA dimer) on the column. Dashed lines indicate expected molar masses for monomeric COMA or dimeric COMA: 142337 g mol−1 or 284674 g mol−1; our experimentally determined molar mass from the principal SEC elution peak: 299100 g mol−1 ± 5683 g mol−1 (mean ± standard deviation from a single SEC-MALS experiment); max. dimeric COMA concentration, from refractive index measurement, in principal peak: 2.4 μM.
Figure 5Diagram for LEC-specific ablation of VEGFR3 in adult mice (VR3iΔLEC). Images and comparison of VEGFR3 expression (presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum between WT and VR3iΔLEC mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm. Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and VR3iΔLEC mice. Note that, in addition to the alterations in the lacteal lengths in VR3iΔLEC mice, diameters of the lacteal also shrank by VEGFR3 ablation. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm. Images and comparisons of number of Prox1+ LECs within initial 100 μm portion in CD31+/LYVE-1+ lacteals of jejunum from WT and VR3iΔLEC mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm. Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum from WT and VR3iΔLEC mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm. Comparisons of serum triglyceride (TG) at indicated time points in WT and VR3iΔLEC mice (n = 6 mice/group). Comparison of protein levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group). Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group).
Nucleosome assembly in vitro and in vivo.Figure 6A Native PAGE gel showing the result of an affinity shift assay scanned to detect AF488- (left) or AF647-labeled DNA (right). 31 nM - 4 µM of yNap1-H2A-H2B (lanes 6-13) were incubated with a mixture of 0.8 µM AF488-labeled H3-H4-DNA tetrasome complexes and 0.8 µM AF647-labeled DNA (lane 5). Lanes 1-3 show migration of tetrasomes (tetra), nucleosomes (nucl) or free H2A-H2B obtained by salt deposition onto AF647-labeled DNA. B Intensity of the nucleosome band (nucl) is plotted. The value at 4 µM of yNap1-H2A-H2B (lane 13) was set to 100%. For non-specific DNA binding, the value in the absence of H2A-H2B (lane 5) was set to 100% intensity and disappearance of the free DNA AF647 band was plotted. Each curve is representative of three independent experiments performed on different days. Standard deviations are shown. C Analysis of MNase-digested chromatin DNA fragments from wild-type (BY4741), nap1Δ, and nap1Δ cells complemented with different yNap1 expression plasmids. Total MNase units per 50 ml of culture are indicated on the bottom of the lanes. Kb+: DNA Marker. D Positional representation of MNase titrated nucleosomal arrays from panel C, comparing with BY4741 (blue traces) with NAP1 deletion (green traces) and other mutants (HBR1-black, HBR2-red, HBR1+2-purple, and IF1-gold traces). Color gradients indicate MNase concentrations (from high to low) and the yellow box represents mononucleosomes. Dashed lines represent nucleosome dyad with respect to BY4741 (WT). Traces are offset vertically for ease of visibility. See also Fig EV4 I.
Intrinsic role of Aub in GSC self-renewal and differentiation (A) Schematic diagram of a germarium showing the somatic cells (blue) and the germline cells (green).Figure 1(A) Schematic diagram of a germarium showing the somatic cells (blue) and the germline cells (green). The spectrosomes and fusomes are shown in orange. The different regions of the germarium are indicated. Region 1: dividing cysts; region 2: selection of the oocyte; region 3: egg chamber with posteriorly localized oocyte. GSCs: germline stem cells; CB: cystoblast. (B-E) Immunostaining of germaria from 7 day-old females with anti-Vasa (green, B-D) or anti-GFP (green, E), and anti-Hts (red). DAPI (blue) was used to visualize DNA. (B) aubHN2/+ was used as a control. (C, D) examples of aubHN2/QC42 germ cell loss and tumor, respectively. (E) Phenotypic rescue of aubHN2/QC42 with UASp-GFP-Aub expressed using nos-Gal4. White arrowheads indicate GSCs; the white arrow indicates GSC loss. (F) Quantification of mutant germaria with 0 to 1 GSC, or with GSC tumors shown in (B-E). The number of scored germaria (n) is indicated on the right graph. *** p-value <0.001, ns: non significant, using the χ2 test. (G-H') Germaria containing control (G, G') or aubHN2 mutant (H, H') clonal GSCs stained with anti-GFP (green) and anti-Hts (red), 14 days after clone induction. DAPI (blue) was used to stain DNA. Clonal cells are marked by the lack of GFP. Clonal GSCs and cysts are outlined with dashed line. White arrowheads show clonal GSCs in the control. aub mutant clonal GSCs have been lost (H, H'). Scale bar: 10 μm in B-E and G-H'. (I) Quantification of germaria containing at least one clonal GSC at 7, 14 and 21 days after clonal induction. (J) Division rate of wild-type and aubHN2 clonal GSCs. The number of scored germaria (n) is indicated. *** p-value <0.001 using the two-tailed Student's t test.
Figure 1A Representative images of Bem1-GFP and the PS marker LactC2-GFP in wild type and cho1∆ cells. Images show average intensity projections of deconvolved z-stacks. Black and white pixels were inverted to improve contrast for display. B Frequency of cells displaying polarized Bem1-GFP signal in wild type and cho1∆ cells. Values are means ± SD, n > 100 cells in each of 3 independent experiments. Paired Student's t-tests were performed. C Imaging of the PI4P probe GFP-2xPHOsh2 in wild type and cho1∆ cells. Images show average intensity projections of deconvolved z-stacks in which black and white pixels were inverted. D Scatter dot plot showing PI4P levels at the plasma membrane (see experimental procedure for details of the quantification) in wild type and cho1∆ cells. Values are means ± SD, n > 100 cells, observed over 3 experiments. Mann-Whitney tests were performed. E Ratio of PI4P average fluorescence intensity in the bud versus mother cell in the cho1∆ mutant. Values are means ± SD, n > 100 cells, observed over 3 experiments. A value > 1 indicates enrichment of the fluorescence in the bud. F Images of Bem1-GFP in cho1∆ 9xMyc-AID-stt4 cells after 30 min treatment with or without 0.5 mM auxin. Images are average intensity projections of deconvolved z-stacks. G Frequency of cells with polarized Bem1-GFP signal or Bem1-GFP in puncta in cho1∆ 9xMyc-AID-stt4 cells treated with or without auxin as shown in F. Values are means ± SD, n > 100 cells in each of 6 independent experiments. Paired Student's t-tests were performed. H Images of Bem1-GFP (cyan) and Cdc24-mCherry (red) signals in cho1∆ 9xMyc-AID-stt4 cells with or without auxin. Images display maximum intensity projections of z-stacks. I Maximum intensity projections of z-stacks displaying Bem1-GFP in the mutants indicated. Bem1-GFP was imaged in mss4-102 cho1∆ cells after shifting to the restrictive temperature for 30 minutes. J Frequency of cells with polarized Bem1-GFP signal after incubation at the time and temperature shown. Values are means ± SD, n > 200 cells in each of 3 independent experiments. Paired Student's t-tests were performed.
Figure 1(A) Dynamic regulation of fluorescently tagged Beta-catenin/Armadillo (Arm-GFP) during mitosis in the growing fly wing epithelium. Notice down-regulation of Arm-GFP as cells round up for mitosis (arrow). Scale bar ~2µm. n>10 independent biological replicates. (B) Dynamic regulation of fluorescently tagged E-cadherin (E-cad-GFP) during mitosis in the growing fly wing epithelium. Notice down-regulation of E-cad-GFP as cells round up for mitosis (arrow). Scale bar ~2µm. n>10 independent biological replicates. (C) E-cad-GFP remains downregulated in cells arrested in mitosis with Colchicine. Scale bar ~2µm. n>10 independent biological replicates. (D) Quantification of the degree of down-regulation of Arm-GFP and E-cad-GFP at the junction between mitotic cells and their interphase neighbours (n > 10 independent samples per cell cycle stage). Mean +/- 1 S.D. shown. (E) Two adjacent mitotic cells show no detectable Arm-GFP at the junction between them. Scale bar ~2µm. (F-I) Electron microscopy cross-sections of wing epithelial cells at interphase, prophase, prometaphase and telophase. Notice adherens junctions (dark staining, highlighted in insets) are downregulated as cells round up for mitosis. Scale bars ~1µm (low mag.) and ~10nm (high mag.). n>10 independent biological replicates. Asterisks indicate a single mitotic cell. Green arrowheads indicate adherens junctions.
Figure 3(A) COUP-TFII expression is significantly increased and co-localized with αSMA+ cells at day 10 of UUO and day 14 of UIRI in mice (n=3 animals per group). Quantification by confocal micrographs in 200x hpf. *p<0.05 **p<0.01 by paired t test; mean ± SD. (B) In a time course of UUO, COUP-TFII expression increased as early as day 2 and preceded the increased expression of αSMA (n=2 animals per time point). Quantification by confocal micrographs in 200x hpf. *p<0.05; ***p<0.001 by two-way ANOVA test; mean ± SD. (C) In the UUO model, COUP-TFII positive cells do not stain with markers of inflammatory cells (T cell marker CD3, neutrophil marker LyG6, and macrophage marker F4/80) (n=3 animals). (D) Using a fate tracing of Gli1+ cells in the UUO model, (E&F) Gli1-tdTomato+ cells expanded primarily in the outer medullary region. In contrast, COUP-TFII+ cells are distributed both in cortex and medulla. A subset of COUP-TFII+ cells (green) overlap with genetically labeled Gli1+ pericyte/perivascular cells (red) in UUO injury model (n=3 animals per group). CTL: contralateral kidney. Quantification by confocal micrographs in 200x hpf.
Mitophagy is restored in ULK1-null MEFs upon reconstitution of the kinase-active form of ULK1 or constitutively active FUNDC1 (S17D)ULK1+/+ and ULK1-/- cells were cultured under hypoxic (1% O2) condition for 12 h or 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1).Figure 3AULK1+/+ and ULK1−/− cells were cultured under hypoxic (1% O2) condition for 12 h or 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted. B ULK1+/+ and ULK1−/− cells were treated with FCCP (20 μM) for the indicated times in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted. C ULK1+/+ and ULK1−/− cells were transfected with FUNDC1−Myc for 24 h and then harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted. D ULK1−/− cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N), then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells lysates were immunoblotted. EULK1−/− cells were transfected with HA−ULK1 or HA−ULK1 and then treated with FCCP (20 μM) for 24 h. Cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted. F FUNDC1‐knockdown MEFs were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17A) for the indicated times. Cell lysates were used for immunoblotting. G ULK1+/+ cells were transfected with FUNDC1‐Myc, and ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D). 24 h after transfection, cells were fixed with 4% paraformaldehyde and stained with anti‐Myc (mouse) and anti‐LC3 (rabbit) primary antibodies for immunofluorescence microscopy. Scale bar, 10 μm. H ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were immunoblotted. I ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were detected with the citrate synthase activity kit. J HeLa cells were transfected with FUNDC1‐Myc, FUNDC1-Myc (S17A), or FUNDC1-Myc (S17D). 24 h after transfection, cell lysates were immunoprecipitated with anti‐LC3 antibody.
Figure 5A) Left panel: representative images of Western Blot of CD36, PPARγ and GAPDH expression of protein extracts of ACM C-MSC treated with scramble siRNA or CD36 siRNA. Right panels: d.a. normalized on the house-keeping protein GAPDH (n=7 biological replicates; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01. B) Left panels: representative images of ORO staining on ACM C-MSC treated with scramble siRNA or CD36 siRNA and cultured in AM with 150µg/ml oxLDL. Right panel: image quantification (n=7 biological replicates; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01. C) Left panel: correlation between CD36/GAPDH and PPARγ/GAPDH d.a. Right panel: correlation between CD36/GAPDH d.a. and ORO lipid accumulation. Regression lines, their equations, R2 and correlation p values are shown (X-Y correlation). Data information: mean ± SEM. * p<0.05; ** p<0.01. D) Left panel: representative images of internalization of oxLDL (red) in ACM C-MSC treated with scramble siRNA or CD36 siRNA, cultured in AM and subjected to 10μg/ml DiI oxLDL treatment. Right panel: quantification of the DiI fluorescence normalized on nuclei number (n=3 biological replicates, arbitrary units; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01. E) Left panel: representative images of internalization of oxLDL (red) in ACM C-MSC cultured in AM or AM+5μM GW9662 and subjected to 10μg/ml DiI oxLDL treatment. Nuclei are counterstained with Hoechst33342 (blue). Right panel: quantification of the DiI fluorescence normalized on nuclei number for each sample (n=3 biological replicates, arbitrary units; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01. F) Left panel: representative images of Western Blot of CD36 and GAPDH expression of protein extracts of ACM C-MSC cultured in AM or AM+5μM GW9662. Right panels: d.a. normalized on the house-keeping GAPDH (n=7 biological replicates; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.