EMBO/sd-character-level-ner huggingface dataset:
Diencephalic astrocytes give rise to progeny in vivoA-E Micrographs of maximum intensity projections (A - D) or single optical section (E) of forebrain sections stained as indicated for fluorescent proteins in astrocytes labelled in 8 mo (A, B, E) and 3 mo (C, D) GLASTCreERT2/Confetti mice at 21 dpt.Figure 7A-E Micrographs of maximum intensity projections (A - D) or single optical section (E) of forebrain sections stained as indicated for fluorescent proteins in astrocytes labelled in 8 mo (A, B, E) and 3 mo (C, D) GLASTCreERT2/Confetti mice at 21 dpt. Note that clusters of duplets (D) and triplets (C) labelled in the same color can be found only in the DIE. Also note that astrocyte somata are often very close together, as also observed after live in vivo imaging of dividing astrocytes Somata of single cells are highlighted in circles. Data information: CTX: cerebral cortex; DIE: diencephalon; HC: hippocampus. Scale bars: 300 µm (A), 100 µm (B), 50 µm (C, D), 20 µm (E).
Figure 6(A) Immunofluorescence staining for the polarized mitochondria (red), Tom20 (blue), and LC3 (green) in Huh7 cells untreated or treated with DFP (1 mM) for 24 h, or with CCCP (30 μM) for 6 h. Boxed areas are enlarged on the right. Scale bar: 10 μm, and 5 μm in enlarged images. (B) Immunofluorescence staining for the polarized mitochondria (red), Tom20 (blue), LC3 (blue), and FTMTD57A (green) using Huh7 cells expressing FTMTD57A. Boxed areas are enlarged on the right. The white puncta in overlaid pictures indicate the colocalization of the polarized mitochondria, Tom20 and FTMTD57A or the colocalization of the polarized mitochondria, FTMTD57A, and LC3. Scale bar: 10 μm, and 5 μm in enlarged images. (C) STAM mice were fed a high-fat diet and orally administered 0.0375 mg/g body weight of DFP, 0.075 mg/g body weight of DFP or distilled water from 4 weeks of age to 16 weeks of age (n=6). Arrows indicate liver tumors. Scale bar: 1.0 cm. The number and maximum size of liver tumors were compared among the three groups. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01. (D) Histology of the nontumorous liver (H&E and Sirius red stain, original magnification x40) and superoxide anion production in the livers of STAM mice fed a high-fat diet with or without DFP (0.075 mg/g) administration. Frozen liver sections of mice were stained with dihydroethidium for assessment of superoxide anion. Scale bar: 100 μm. (E) Histology of the hepatocellular carcinoma using H&E staining in STAM mice fed a high-fat diet. The boxed area is enlarged on the right (x40 in left panel and x400 in right panel). The tumor has increased cell density, an increased ratio of nucleus to cytoplasm, and thickened trabeculae Scale bar: 100 μm. (F) Electron microscopic images of livers in control (C57BL/6) and DMBA + HFD mice that were orally administered 0.075 mg/g of DFP or distilled water. Scale bars: 2.0 μm. The boxed areas (x 1,500) are enlarged below (x6,000 on left and x10,000 on right). The white arrow indicates mitophagosome-like structures. The mean number of mitophagosome-like structures/100 μm2 was compared for four randomly selected areas (technical replicates) among the four groups (control, DMBA + HFD mice treated with 0, 0.0375 or 0.075 mg/g of DFP) (n=6). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05.
Capsid variants of a random AAV display peptide library during five rounds of in vivo selection enriching for brain-homing library particles.Figure 1A Randomly chosen library peptide inserts (shown in single-letter code) recovered from the brain. Ten clones were sequenced after each round of selection. Intravenously injected library particles were allowed to circulate for two days in each selection round (n = 1 animal /selection round, age 8-12 weeks). Five selection rounds were performed. Amino acids characterizing the emerging motifs are highlighted in different colors. The consensus motif of each selection round is shown at the bottom of each column. Sequences were considered to show a consensus if at least four different clones displayed the same amino acids at the same position or at two adjacent positions. B In vivo transduction profile of luciferase reporter vectors displaying variants of the enriched library capsid sequence motifs or unmodified wild type AAV2 control capsid. Four weeks after i.v. injection of 5x1010 genomic particles/mouse containing a CMV-luciferase reporter gene, luminescence was measured in the brain and control organs. Data are shown as bars (mean) with plotted individual data points (n = 3 animals/group, age 8-12 weeks).
Figure 2A. Huh7 cells were co-transfected with shRNAs fulfilling all requirements for incorporation into the expression strategies from Figure 1C and the HBV expression plasmid pCH-HBV1.3, and HBeAg and HBsAg were quantified in the supernatant 48 h later. An empty AAV vector plasmid was used as control. Another negative control were mock-transfected cells. B. The same experiment was repeated with plasmid pCH-9-3091, which contains a 1.1-fold over-length HBV genome. HBV DNA in supernatant was measured at day four via PCR, using primers that preferentially amplify HBV rcDNA rather than the 1.1-fold over-length HBV sequence contained in the plasmid. C. RT-qPCR results from cell lysates (same experiment as in panel A) with primers specific for the 3.5 kb HBV transcripts (consisting of pregenomic [pg] RNA and the precore transcript; left), or with primers that detect all HBV transcripts (right). D. Southern blot analysis of lysates obtained 72 h post-transfection of HepG2 cells treated with the same plasmids as in panel A.
Figure 4A. HeLa cells stably expressing CHMP4B-eGFP were treated with 250 μM LLOMe and 75 nM Lysotracker DND-99 and fixed at different time points as indicated. After 10 min of LLOMe treatment CHMP4B is recruited whereas the number of Lysotracker positive foci is reduced. After 30 min, lysosomes gain back functionality (judging by the increased number of Lysotracker foci) and appear recovered after 1 h indicating that the ESCRT complex is able to seal the damaged endolysosomal membranes. Representative confocal images for each time point are shown. Scale bars: 5 μm B. Quantification graph (>250 cells per condition from four independent experiments) showing CHMP4B and Lysotracker positive foci per cell at different time points. Error bars correspond to 95% confidence intervals C. Quantification graph showing dynamics of Lysotracker recovery in control (siCtrl) and both TSG101 and ALIX depleted cells. HeLa cells stably expressing CHMP4B-eGFP were co-transfected with siRNAs against TSG101 and ALIX. 48 h post-transfection, cells were pre-treated for 20 min with 75 nM Lysotracker Deep-Red, which was used as a read-out. While the decrease in the number of Lysotracker spots is quickly recovered in siCtrl, simultaneous depletion of ALIX and TSG101 leads to a severe impairment in lysosomal repair. Data from >40 cells per condition from four independent live-cell imaging experiments are shown. Graph is normalized to the area occupied by the cells. Error bars correspond to 95% confidence intervals.
pat1 mutants have a serrated leaf, semi‐dwarf phenotypePlants photographed at 4 weeks of growth in short day conditions, genotypes as indicated.Figure 3pat1 mutants have a serrated leaf, semi‐dwarf phenotypePlants photographed at 4 weeks of growth in short day conditions, genotypes as indicated. Col‐0 and eds1‐2 plants and pat1‐1 and pat1‐1/eds1‐2, respectively, were placed in the middle of the same pots before the pictures were taken.
Figure 1A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm. B. Quantification of the number of CETN3 foci per spermatocyte (n = 30) at each meiotic prophase substage. C. Representative images of unseparated (< 1 μm apart), initial separation (1-5 μm apart), intermediate separation (5-11 μm apart), and pole separation (>11 μm apart) of centrosomes. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm. . Centrosome separation was scored in n = 30 spermatocytes at each prophase substage using the four centrosome separation classifications P value obtained from a Kruskal-Wallis one-way ANOVA. **P<0.01. E. Spermatocytes undergoing meiosis I through meiosis II were immunolabeled against PCNT (purple), CETN3 (green), and SYCP3 (red), and stained with DAPI (blue). Zoomed images of centrosomes below each panel. Scale bars = 5 μm. F. Model for centriole duplication and centrosome separation patterns throughout the meiotic prophase and division stages. During the leptonema to zygonema transition the first round of centriole duplication occurs. As cells progress through meiotic prophase I, centrosomes mature and acquire distal appendages. During diakinesis centriole separation occurs, allowing the formation of a bipolar spindle during the first meiotic division. During interkinesis centrioles duplicate a second time and separate to form a bipolar spindle at meiosis II.
a–d) Rotenone (Rot) increased the number of GFP–LC3 puncta and the level of co-localization with mitochondria (arrows) in SH-SY5Y cells (a–c; 1 μM) and primary cortical neurons (d; 250 nM), quantified in Fig.figf1(a-d) Rotenone (Rot) increased the number of GFP-LC3 puncta and the level of co-localization with mitochondria (arrows) in SH-SY5Y cells (a-c; 1 μM) and primary cortical neurons (d; 250 nM), quantified in Fig. 3f and Supplementary Fig. S1b,c. Veh, vehicle. (e,f) Rotenone increased delivery of MitoTracker Green-stained mitochondria (mt) to LysoTracker Red (LTR)-stained lysosomes, inhibited by siRNA knockdown of ATG7 or LC3 in SH-SY5Y cells. Ctrl, control. Right: RNAi knockdown. (g) Rotenone decreased IMM (COXIV), OMM (TOM40) and matrix (MnSOD) protein expression levels in primary neurons, reversed by bafilomycin (Baf) inhibition of autolysosomal degradation, quantified in Supplementary Fig. S1e. Mean ± s.d. of n = 7 independent experiments for b,c, and n = 3 independent experiments for f,g (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle control; †P0.05 versus Rot/control siRNA. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.
Figure 6A, Experimental protocol followed for the differentiation of cardiomyocytes from iPSCs in the absence or presence of miR-203 mimics and Dnmt3a/b cDNAs. B, Representative immunofluorescences showing cardiac Troponin T (cTnT, green) and nuclei (Hoechst, blue) staining of in vitro-generated cardiomyocytes derived from wild-type iPSCs transiently transfected with either control mimics, miR-203 mimics or miR-203 mimics + Dnmt3a/b cDNAs. Pictures were taken at day 15 of differentiation. Scale bars, 68 μm. The cTnT-positive area in these cardiomyocytes is shown in the right histogram. Data are represented as mean ± s.d. (n=2 independent experiments with 6 replicates each). C, Beating frequency (measured as number of beats per 5 seconds) of these cardiomyocytes at day 15 of differentiation. Data are represented as mean ± s.d. (n=8 independent replicates). D, miRNA or mRNA levels as determined by quantitative PCR of the indicated transcripts at different time points during cardiomyocyte differentiation in the indicated samples. Data are represented as mean ± s.d. (n=2 independent experiments with 6 replicates each).
Figure 7A. HeLa cells were transfected with different siRNAs as indicated. 48 h post-transfection cells were treated with 250 μM LLOMe for 3, 6, 10 hours harvested and stained for Annexin V. Cells were immediately processed for flow cytometry. Already after 3 h of treatment with LLOMe, cells co-transfected with TSG101+ALIX siRNA showed an increase in apoptotic cell death, whereas TSG101 siRNA transfected cells showed elevated Annexin V staining after 6 h of treatment Data information: Graphs show average data from a set of three independent experiments where >10,000 cells per condition were analyzed. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. p-values are presented where significant. *p-value≤ 0.05, **p-value≤ 0.01 and ***p-value≤ 0.001 B. Experimental set up as in (A) with additional propidium iodide (PI) staining of transfected HeLa cells Data information: Graphs show average data from a set of three independent experiments where >10,000 cells per condition were analyzed. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. p-values are presented where significant. *p-value≤ 0.05, **p-value≤ 0.01 and ***p-value≤ 0.001
| DND1 interacts directly with NANOS2 via its zinc finger motif.Figure 1A, B, Western blotting analysis of proteins co-precipitated with anti-FLAG antibody from testis extracts of E15.5 wild-type embryos and transgenic embryos expressing FLAG-tagged NANOS2 with or without RNase (A) or from extracts of HeLa cells transfected with FLAG-tagged DND1 and with or without HA-tagged NANOS2 (B). Precipitates were analyzed with the indicated antibodies. C, GST pull-down assay using E. coli extracts expressing GST, GST-fused NANOS2, or NANOS3 mixed with MBP-tagged LacZ or DND1. Precipitates were analyzed by Coomassie Brilliant Blue (CBB) staining or western blotting with an anti-MBP antibody. An arrowhead indicates MBP-DND1 co-precipitated with GST-tagged NANOS2 or NANOS3. D, E, Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with FLAG-tagged NANOS2, NANOS2 (ΔN10), NANOS2 (ΔC10), NANOS2 (C61A, C96A) or NANOS3, and HA-tagged DND1 (D) D, E, Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with FLAG-tagged NANOS2, NANOS2 (ΔN10), NANOS2 (ΔC10), NANOS2 (C61A, C96A) or NANOS3, and HA-tagged DND1 (D) or with FLAG-tagged NANOS2, NANOS2 (C61A), NANOS2 (C96A) or NANOS2 (C61A, C96A), and HA-tagged DND1 (E). Precipitates were analyzed with the indicated antibodies. F, GST pull-down assay using E. coli extracts expressing GST-fused DND1 mixed with MBP-tagged LacZ, NANOS2, NANOS2 (C61A, C96A) or NANOS2 (60-114). Arrowheads indicate MBP-tagged NANOS2 (lane 2) or NANOS2 (60-114) (lane 4).
a) Midguts dissected from animals expressing Uba1IR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast (DIC) microscopy.figf6(a) Midguts dissected from animals expressing Uba1IR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown. (b) Cell size quantification (μm2) from a; n = 16 animal intestines per genotype with 1-5 cells measured per intestine. (c) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function mutant cell clones (lacking RFP) and analysed by fluorescence and differential interference contrast microscopy. Wild-type (+/+) control cells possess stronger RFP and heterozygous Uba1H33/wild-type (H33/+) cells have weaker RFP. Representative images are shown. (d) Quantification (μm2) from c, n = 8 (+/+), n = 11 (H33/+) and n = 11 (H33/H33) animal intestines per genotype with 1-5 cells measured per intestine. (e) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function MARCM mutant cell clones (GFP-positive) that also have mCherry-Atg8a expressed in all cells and analysed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown. (f) Midguts expressing GFP-Atg5 in enterocytes (larger nuclei), and with an Uba1H33 loss-of-function clone cell (lacking RFP) at puparium formation analysed by fluorescence microscopy. Representative images are shown. (g) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function MARCM mutant cell clones (GFP-positive) that are stained with p62 antibody and analysed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown. (h) Midguts dissected from animals expressing Dts7, a dominant temperature-sensitive mutant of the β2 subunit of the proteasome, specifically in GFP-marked cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (i) Cell size quantification (μm2) from h, n = 6 animal intestines per genotype with 1-5 cells measured per intestine. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.
Figure 3(A) Transfections of HEK293 cells revealed that both wild-type GFP-PI4KB and RL494EA GFP-PI4KB localize to the Golgi. (B) WT c10orf76 also localized to the Golgi, however, the PI4KB binding deficient mutant of c10orf76 (FLH409AAA) predominantly localized to the cytosol. (C) Cartoon schematic of rapamycin-inducible mitochondria recruitment. The AKAP1-FRB-CFP construct is localized to the outer mitochondrial membrane, while the mRFP-FKBP12-PI4KB and eGFP-c10orf76 are localized in the Golgi as well as within the cytoplasm where they can form a complex. Upon addition of rapamycin, the mRFP-FKBP12-PI4KB construct is translocated to the mitochondria. (D) Mitochondria recruitment experiment with wild-type PI4KB and c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after rapamycin (100 nM) treatment (bottom). Middle: mRFP-FKBP12-PI4KB at high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 at a high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). (E) Schematic of the rapamycin-inducible mitochondria recruitment experiment with mutant PI4KB and WT c10orf76. (F) Mitochondria recruitment experiment with mutant PI4KB and WT c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after (bottom) rapamycin treatment. Middle: mRFP-FKBP12-PI4KB(RL494EA) is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 is located in the cytosol before (top) and after (bottom) rapamycin induction.
Figure 4A Plots of relative mRNA abundance of Nipped-A in whole head extracts of w1118 flies in LD and DD1 determined by qRT-PCR (n=3). B Left panel: Western blots of proteins from whole heads of w1118 and Nipped-A RNAi flies collected during LD or DD1 and probed with NIPPED-A antibody. Right panel: quantification of NIPPED-A protein levels of blots in the left panel (LD, n=3; DD1, n=5). C Chromatin immunoprecipitation (ChIP) assays to detect NIPPED-A binding at E-box, transcription start site (TSS) and gene body of tim, Pdp1ε and per using w1118 flies (n=3). Data information: Error bars represent SEM. Two-way ANOVA, significant effect between NIPPED-A and IgG were found for tim E-box (P<0.001), Pdp1ε E-box (P<0.001), per E-box (P<0.001), tim TSS (P<0.001), Pdp1ε TSS (P<0.001), per TSS (P<0.001), tim gene body (P<0.001), Pdp1ε gene body and per gene body (P<0.001). Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. G4, GAL4; U, UAS.
Figure 5(A-B) Plasma concentrations of NfL (A) and pNfH (B) were measured in the 304Q knock-in SCA3 mouse model. Heterozygous animals become symptomatic at ≈8 months of age. (C-F) Tissue levels of soluble and aggregated mutant ataxin-3 were measured in cerebellum (C, E) and frontal lobe (D, F).
Figure 6The muscle-specific (A-B) Mef2-GAL4 drivers were crossed to UAS-RNAi lines to knockdown the expression of the gene markers for the myoblast clusters. (A, Viability test quantified by scoring the percentage of viable animals at each developmental stage, including early pupa (black), pharate (grey) and adult (white), relative to the total number of animals. Data are expressed as stacked bars showing the median with interquartile range (n≥ 3 independent biological replicates), unless noted as viable (NQ) and lethal (NQ), which stands for animals that were viable and lethal, respectively, but no quantitative analysis were performed. (B, Flight ability scored by quantifying the percentage of flies landing on each section of the column (top, middle and bottom) as indicated on the diagram on the right. Data are expressed as stacked bars. The total number of flies used in each assay was pooled together and displayed next to each bar (N≥6). n/a stands for non-applicable since animals are not viable. The muscle-specific 151-GAL4 drivers were crossed to UAS-RNAi lines to knockdown the expression of the gene markers for the myoblast clusters. C). Viability test quantified by scoring the percentage of viable animals at each developmental stage, including early pupa (black), pharate (grey) and adult (white), relative to the total number of animals. Data are expressed as stacked bars showing the median with interquartile range (n≥ 3 independent biological replicates), unless noted as viable (NQ) and lethal (NQ), which stands for animals that were viable and lethal, respectively, but no quantitative analysis were performed. D). Flight ability scored by quantifying the percentage of flies landing on each section of the column (top, middle and bottom) as indicated on the diagram on the right. Data are expressed as stacked bars. The total number of flies used in each assay was pooled together and displayed next to each bar (N≥6). n/a stands for non-applicable since animals are not viable. E. Confocal single plane images of third instar larval wing discs from Mef2>mCherry-RNAi, (left panel) and Mef2>chinmo[HM04048]-RNAi (right panel) stained with anti-Zfh1 (red) and DAPI (cyan). Scale bars: 50 μm. F. Quantification of phenotype displayed in (E). Stacked bars showing the percentage of wing discs scored as either normal or as abnormal. Data are expressed as mean ± sem, N = 32 discs per genotype, n = 3 independent experiments. G. Quantification of viability test. Since the UAS-Ama-RNAi[GD12733] transgene is inserted on Chromosome X the genotype of females is Mef2>Ama-RNAi whereas males are Mef2>+. Box plot showing the ratio of flies with genotype of interest (females) over control flies (males). The whiskers show 5-95 percentile, and the middle line the median. * p=0.014 (Mann-Whitney test, N≥6 independent biological replicates). Full genotypes are w, UAS-Dicer2;+; mCherry-RNAi/Mef2-GAL4 and w, UAS-Dicer2, UAS-Ama-RNAi[GD12733];+; Mef2-GAL4.
Figure 5A. 293T cells were transfected with the indicated siRNAs, cultured for 24 hours, and then transfected with the indicated DNAs. 48 hours after siRNA transfection, the cells were harvested and ubiquitination of Flag-AMOTL2 was examined. C, control siRNA. B. 293T cells were transfected with the indicated combinations of DNAs, cultured for 24 hours, and subjected to in vivo ubiquitination assays. C. RPE cells transduced with vector (control) or Flag-AMOTL2 were seeded under sparse (S) or dense (D) conditions, and an in vivo ubiquitination assay was performed. D. RPE cells transduced with Flag-AMOTL2 were transfected with a 1:1 mixture of the two USP9X siRNAs. One day after siRNA transfection, the cells were re-seeded to the sparse condition and an in vivo ubiquitination assay was performed after one day. E. 293T cells were transfected with the indicated combinations of DNAs, and a co-immunoprecipitation assay was performed. F. Sparsely cultured RPE or MCF10A cells were immunoprecipitated with an anti-AMOTL2 antibody.