EMBO/sd-character-level-ner huggingface dataset:
Figure 2Western blot showing siRNA-mediated depletion of FOXA1 and/or FOXA2 in the low-grade CFPAC1 cell line. The three biological replicates used in the subsequent experiments are shown. Heatmap showing row-normalized expression of differentially expressed genes in CFPAC1 cells depleted of FOXA1/2 (FDR ≤ 0.05 and absolute log2 FC ≥ 1). Blue and orange depict down- and up-regulation, respectively. FOXA2 and FOXA1 expression in wild type and genome-edited high-grade PANC1 cells. Three different bulk populations were used for RNA-seq experiments. Heatmap showing row-normalized expression of differentially expressed genes in PANC1 cells deleted of FOXA2 (FDR ≤ 0.05 and absolute log2 FC ≥ 1). Blue and orange depict down- and up-regulation, respectively. Venn diagram showing the overlap between FOXA1/2-regulated genes in CFPAC1 and PANC1 cells. Representative Gene Ontology (GO) terms enriched in genes differentially expressed upon FOXA1/2 depletion in CFPAC1 cells. Terms are ranked by the negative log10 p-value from the hypergeometric test. Panels on the right show the effects of FOXA1/2 depletion on cell morphology and adhesion to multiple substrates. Three replicates per condition were used: circularity index was measured on 100 different cells per condition while adhesion is reported as mean of three different fields per replicate for a total of nine observations per condition. Representative GO terms enriched in genes differentially expressed upon FOXA2 deletion in PANC1 cells. Terms are ranked by the negative log10 p-value from the hypergeometric test. Panels on the right show the effects of FOXA2 deletion on cell morphology and adhesion to multiple substrates. Three different bulk populations were used as biological replicates: circularity index was measured on 60 different cells per bulk population while adhesion is reported as mean of six different fields per bulk for a total of eighteen observations per condition.
Figure 44-month-old APP/PS1 (n = 13, male) and age-matched wild-type (Wt, n = 14, male) mice were intravenously injected with Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for 12 weeks. After the completion of behavioural tests, the brains of each group of mice were subjected to analysis.A. ThS-stained insoluble Aβplaques in whole brain and the hippocampal region of each group. Scale bars = 1 mM (upper), 200 μM (lower).B-G. Statistics of ThS-positive Aβplaques in the brains of APP/PS1mice. Total numbers and areas of ThS-positive Aβplaques in the whole brains (B and C), cortex (D and E) and hippocampus (F and G).
Figure 6A, B Pull-down assay. Total proteins prepared from EGR2-Myc or EGR2G2A-Myc transgenic plants was immunoprecipated with anti-Myc agarose beads, and then incubated with GST-OST1. Anti-Myc and anti-GST antibody were used to detect GST-OST1 and EGE2-Myc. Representative pictures are shown in (A). Relative interaction is shown in (B). Data information: In (A Relative band intensities are shown. In (B each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test). C LCI of EGR2 and EGR2G2A with OST1 in N. benthamiana leaves. EGR2-cLuc/OST1-nLuc and EGR2G2A-cLuc/OST1-nLuc were expressed in N. benthamiana leaves for 48 h. EGR2-cLuc/nLuc, EGR2G2A-cLuc/nLuc, and cLuc/OST1-nLuc were used as negative controls. Scale bar: 2 cm. D, E Co-IP assay. EGR2-Myc/HF-OST1, EGR2G2A-Myc/HF-OST1, HF/EGR2-Myc and HF/EGR2G2A-Myc were expressed in N. benthamiana leaves. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-HA and anti-Myc antibodies were used to detect HF-OST1, EGR2-Myc and EGR2G2A-Myc, respectively. Representative pictures are shown in (D), and relative interaction is shown in (E). Data information: In : Relative band intensities are shown. In each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test). F, G Effect of EGR2G2A on the interaction of OST1 and EGR2 in Arabidopsis protoplasts expressing HF-OST1/EGR2-GFP/EGR2-Myc, HF-OST1/EGR2-GFP/EGR2G2A-Myc, or HF-OST1/EGR2-GFP. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-Myc, anti-GFP, and anti-HA antibodies were used to detect EGR2-Myc, EGR2-GFP, and HF-OST1, respectively. Representative pictures are shown in (F), and relative interaction is shown in (G). Data information In Relative band intensities are shown. In each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test). H, I Effect of cold stress on the interaction of OST1 and EGR2. Arabidopsis protoplasts expressing EGR2-Myc/HF-OST1 and EGR2-Myc/HF were treated at 4°C for 0 and 2 h. Total proteins were immunoprecipated by anti-HA agarose beads. The co-immunoprecipitated products were detected by anti-HA and anti-Myc antibodies, respectively. Representative pictures are shown in (H), and relative interaction is shown in (I). Data information: In H) Relative band intensities are shown. In I): each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).
Figure 4Two-photon images of microglia (e-GFP) cells clustering around amyloid plaque for APP and APP/Nlrp3-/- (5 and 15 months old). Scale bar: 20µm b) Quantification of morphological parameters in (a) (mean of 5-6±SEM). Flow cytometry plots from APP and APP/Nlrp3-/- mice (15 months old), cells were gated on CD11b and MXO4 after microglia isolation. d) Relative Aβ microglia uptake quantification
Figure 3A. Absolute quantitation of 24, 25 and 27-hydroxycholesterol in primitive and metastatic melanoma cells as indicated. (T-test analysis, ***p-value<0.001, N=3 biological replicates, data are mean ±SD). B. Histogram representing R square of correlation analysis between absolute quantitation of 24, 25 and 27-hydroxycholesterol and label free quantitation of APOE in a cohort of primitive (A375, IPC298) and metastatic (SKMEL5, SKMEL28) melanoma cell lines. C. Confocal fluorescence images of anti-HMB45 PMEL antibody signal (green) and DAPI staining (blue) in IGR37 and IGR39. Scale bar is 10µm. D. Western blot on IGR37 and 39 cellular lysates probed with anti-HMB45 PMEL antibody and anti HDAC2 to check the loading of similar amount of total lysates.
RNA-seq of astrocytes from adult mouse cerebral cortex grey matter and diencephalonA Schematic drawing of Ribotag assisted mRNA isolation from astrocytes isolated from regions dissected as indicated in red (CTX GM) and blue (DIE).Figure 8A Schematic drawing of Ribotag assisted mRNA isolation from astrocytes isolated from regions dissected as indicated in red (CTX GM) and blue (DIE). B Micrographs of forebrain sections of 3 mo old GLASTCreERT2/RPL22HA/eGFP 21 dpt mice stained for HA show maximum intensity projections of HA+ expressing cells with astroglial morphology. CTX GM: cerebral cortex grey matter; DIE: diencephalon. Scale bars: 100 µm. C Micrographs of forebrain sections of 3 mo old GLASTCreERT2/RPL22HA/eGFP mice 21dpt stained for HA and S100β showing the specificity of the mouse line with HA detected only in S100β+ astrocytes. DIE: diencephalon. Scale bar: 70 μm. Data information: CTX GM: cerebral cortex grey matter; DIE: diencephalon; n=3 biological replicates, tissue from 3 mice was pooled D Histogram showing expression levels of the top 10 astrocyte specific markers genes in DIE (blue bars) and CTX GM (red bars) samples. Data information: CTX GM: cerebral cortex grey matter; DIE: diencephalon; n=3 biological replicates, tissue from 3 mice was pooled E Principal Component Analysis (PCA) from DeSeq2 expression data shows distinct clusters of samples from 3 DIE (blue) and 3 CTX GM (red) astrocyte sample preparations. F Venn diagram depicting co-expressed and differentially expressed gene numbers in DIE and CTX GM astrocytes G, H Heatmaps depicting top 40 differentially expressed genes (G, row scaled log normalized read counts) or transcription factors (H, Library size normalized log2 transformed read counts; from DIE and CTX GM astrocytes. Transcription factors were identified according to the content of AnimalTFDB database (http://www.bioguo.org/AnimalTFDB/). I Bar chart depicting significantly enriched GO terms (analyzed first by Gorilla followed by Revigo) based on the 379 differentially expressed transcription factor genes (DeSeq2, cutoff pAdj0.01) comparing DIE and CTX GM astrocytes. Terms related to cell division, cell proliferation etc. are highlighted in red. (P-value threshold was set to 0.01
NSCs retained the original genotype and sequence.Figure 4A: Quantification of gene expression for NSC markers PAX6, NESTIN and SOX2 for all NSCs from RT-qPCR analysis. The expression of the neural stem cell markers is assessed with fold change using the comparative ΔΔCt method by normalizing NSCs to iPSCs (n=3, technical replicates per ESC line or iPSC clone). B, C: Quantification of protein expression level for PAX6 (B, n=6, technical replicates per line for ESCs; n=7, technical replicates per clone for control; n=8, technical replicates per clone for WS5A; n=9, technical replicates per clone for CP2A) and NESTIN (C, n=6, technical replicates per line for ESCs; n=7, technical replicates per clone for control; n=8, technical replicates per clone for WS5A; n=9, technical replicates per clone for CP2A) for iPSC-derived NSCs using flow cytometry. D: Sequencing chromatogram showing the homozygous c.2243G>C variation in POLG in WS5A iPSC-derived NSCs and the heterozygous c.1399G>A and c.2243G>C variation in POLG in CP2A iPSC-derived NSCs. E: Representative confocal images showing glial and neuronal lineages derived from NSCs. a, Immunostaining of NSC-derived astrocytes with GFAP (red) staining (scale bar, 50 µm). b, Immunostaining of oligodendrocytes showing GALC (red) positive labeling (scale bar, 50 µm). c, Dopaminergic neurons showing TH (green) and TUJ1 (red) positive staining (scale bar, 25 µm). Nuclei are stained with DAPI (blue).
Figure 5Mobility shift assay with end-labeled dgcM RNA (210 nt species from +1) in the presence or absence of unlabeled OmrA or OmrB. When indicated, single-stranded DNA oligos were pre-annealed to site 1 (dgcM_site1), site 2 (dgcM_site2), or both, before addition of sRNAs. The additions for each sample are indicated above the Figure, and the identities of the nucleic acid complexes are indicated on the left for guidance.
Figure 3A. M-MØP (blue: P522; red: R522) were loaded with Fluo-8 Ca2+ indicator and examined for peak changes in fluoresce after exposure to LPS (50 ng/ml) or Aβ1-42 oligomers (0.5 µM). Data information: Data shown as the mean±SD of 3 independent experiments. The data were analysed by two-way ANOVA with Sidak's post-tests (*=p<0.05, **=p<0.01, ***=p<0.001). B. Microglia from Plcg2P522 (blue: P522) mice and Plcg2R522 mice (red: R522) from neonate cortex (solid colour) or hippocampus (striped) were loaded with Fluo-8 Ca2+ indicator. These cells were then examined for peak changes in fluoresce after exposure to LPS (50 ng/ml) or Aβ1-42 oligomers (0.5 µM). Data information: Data shown as the mean±SD of 3 independent experiments. The data were analysed by two-way ANOVA with Sidak's post-tests (*=p<0.05, **=p<0.01, ***=p<0.001). C-E. M-MOPS (C), primary mouse microglia (D) and hIPSC (E) (blue: P522 and red: R522) were loaded with Fluo-8 Ca2+ indicator and examined for peak changes in fluoresce after exposure to DOPS-liposomes (25 μg/ml). Data information: Data shown as the mean±SD of 3 independent experiments. The data were analysed by two-way ANOVA with Sidak's post-tests (*=p<0.05, **=p<0.01, ***=p<0.001).
Regulatory connections present in model.Figure 1Regulatory connections present in modelOur previous model of the photoperiod pathway (inset top; Song et al, 2012) included the circadian clock model of Locke et al (2005) (orange, inset top). The new model includes the circadian clock model of Pokhilko et al (2012) (orange, inset bottom). Circadian clock components are represented in red, flowering time pathway components in green and hypocotyl elongation pathway components in blue. Dashed lines represent forms of regulation that have been modelled previously; solid lines represent new regulatory connections. Protein components are represented by rectangles and mRNA by parallelograms. Model components are denoted by their abbreviated names (see text). PIF-interacting proteins (e.g. DELLAs, PAR1, HFR1) are designated "Interactors" in the hypocotyl elongation pathway.
Figure 4A) Analysis of RNA-seq results focusing on transcripts of genes containing at least one minor intron. Genes were divided into two categories depending on whether they were differentially expressed upon FUS KD (DEG) or whether their expression was not affected (non-DEG). The percentage of genes with at least one minor intron is depicted for both categories. Compared to the non-DEG, the % of minor intron containing genes is significantly enriched in the DEG. Effect size: 1.65, p-value: 3.49e-10. B) Histogram of differentially expressed minor intron-containing genes. Genes are grouped according to the level of differential expression. Approximately two thirds of the affected genes are downregulated in the absence of FUS. C) Bar plot showing the percentage of differentially spliced introns among all expressed introns. Major and minor introns are displayed separately. More than 30 % of minor introns are differentially spliced under FUS knockout, whereas only a small subset of the major introns is affected. D) The cumulative plot shows the abundance of minor splice sites among the most differentially expressed splice sites in the RNA-seq data from the FUS KO SH-SY5Y cells. The most differentially expressed sites (ranked from left to right) are depicted on the x-axis, while the y-axis shows the number of minor splice sites among them. The black line represents the distribution observed in the RNA-seq data. This distribution was compared to what would be expected if minor and major splice sites were equally affected by the FUS knockout. This hypothetical distribution (shown in green) was computed with a hypergeometric function. This analysis reveals a high enrichment for minor splice sites, indicating that the usage of minor splice sites is more often altered upon FUS KO relative to the usage of major splice sites.
Novel capsids outperform AAV2 and AAV2.Figure 2Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2-month-old mice. (A) In vivo cSLO examinations of the mouse retina at 1-, 2- and 3- weeks post injection (WPI) of 8E9 total vg of ss-hRho-eGFP packaged with parental serotype AAV2, state-of-art AAV2.7m8, novel AAV2.GL and AAV2.NN capsid variants. (B) qRT-PCR data comparing fold change in eGFP transcript levels relative to ITR2 amplicons from retinal samples infected with all four capsid variants. (C) Confocal scans of retinal cross sections, with focus on the ONL, immunolabelled for DAPI, eGFP and recoverin. The upper panel shows only the DAPI and eGFP signal in the ONL and the lower panel shows the same images (noted with asterisk *) with merged DAPI, eGFP and recoverin signal. Acquisition settings were kept constant for all samples. Scale bar: 50 μm. ONL, outer nuclear layer.
miR-511 is a genuine regulator of TNFR1 and has therapeutic potential.Figure 4Inhibition of Rluc activity of the psiCHECK-Tnfrsf1a-3′UTR reporter plasmid with B sequence (black, left panel) and S sequence (grey, right panel) by miR-511 transfection in HEK-293T cells (n = 7-8). TNFR1 protein levels in B MEF cultures (black, n = 18) and S MEF cultures (grey, n = 24) 24 h after transfection with miR-511 or miR-CTR. TNFR1 protein levels were measured in cell lysates by ELISA. TNFR1 protein levels in B MEF cultures (black, n = 18) and S MEF cultures (grey, n = 18) 24 h after transfection with anti-miR-511 or anti-miR-CTR. TNFR1 protein levels were measured in cell lysates by ELISA. In vivo effect 24 h after hydrodynamic injection of B mice with plasmids expressing miR-511 (grey, n = 19), miR-CTR (black, n = 19) or PBS (black, n = 8). Liver TNFR1 protein levels (left panel) were measured by ELISA. Twenty-four hours after plasmid injection, mice were injected with 25 μg TNF. All mice pretreated with miR-511 (grey, n = 12) survived, while 50% of the mice pretreated with miR-CTR (■, black, n = 12) or PBS (●, black, n = 12) died from TNF injection (middle panel). Twenty-four hours after injection of TNF, body temperatures of mice pretreated with miR-511 were significantly higher than those of the miR-CTR and PBS groups (right panel) (all groups n = 10). Survival of B mice and TNFR1−/− mice injected with LPS, 24 h after miR-511 or miR-CTR hydrodynamic plasmid injection. B mice pretreated with miR-511 (grey, n = 16) were significantly protected against 200 μg LPS compared to miR-CTR-pretreated mice (■, n = 15) or PBS-pretreated mice (●, n = 15) (left panel). No difference in survival was found between TNFR1−/− mice pretreated with miR-511 (grey, n = 11) or with miR-CTR (■, n = 9) against 500 μg LPS (right panel). Effect of hydrodynamic injection of plasmids expressing miR-511, miR-CTR or PBS on ConA-induced hepatitis in B mice (all groups n = 10). A total of 360 μg ConA was injected i.v. 24 h after plasmids and 8 h later mice were analysed: liver damage (serum ALT levels, left panel); body temperatures of mice 8 h after ConA injection (middle panel); TNF levels (right panel) 2 h after injection of ConA revealed similar concentrations of TNF in the three groups. miR-511-injected mice were significantly protected in the ConA model.
Figure 4(A) Hair follicles exhibit immunostaining signal for DNA damage marker H2A.X (red) and apoptosis marker Caspase3 (green) as early as one day after Gata6 iKO induction. Top panel shows a section of UV irradiated epidermis as a positive control for DNA damage. Note that Caspase 3 is not expressed in the H2A.X cells of the epidermis but is found in some iKO hair follicle matrix cells. Right panels show single color images as indicated at top (B) Quantification (average SD) of the percentage of apoptotic cells per matrix as determined by counting of Caspase3+ cells (n=50 follicles from 3 mice per group) (C) Quantification (average SD) of the percentage of DNA damaged cells per matrix as determined by counting of H2A.X+Caspase3- cells (n=50 follicles from 3 mice per group). (D) Regional distribution of apoptosis was analyzed by dividing the matrix into four compartments as shown in the legend (left) and counting the fraction of Caspase3+ cells per each matrix compartments (right; n=50 follicles from 3 mice per group). (E) Proliferation in the matrix compartments shown in (D) was assessed by BrdU labeling for 1 hour, followed by staining, and quantification (F) (interior cells labeled with solid bars, exterior cells labeled with striped bars). Unpaired t-test: *P-value < 0.05. n=20 follicles from 2 mice per group.
Cx43 facilitates the delivery of intraluminal RNAs to target cells.Figure 7A. Equal amounts of EVCx43+ and EVCx43- labelled with SYTO® RNASelect™ green fluorescent stain were incubated with HEK293Cx43+ cells for 30 min, at 37ºC. Scale bars 10 μm (n=5 biological replicates). p-values were derived by Mann-Whitney test. B. Working model for the role of Cx43 in mediating the selective sorting of miRNAs into EV. Cx43 preferentially binds to miRNAs harboring a stable secondary structure, mediating its selective incorporation into EV. Although hnRNPs do not seem to directly participate in Cx43-dependent loading of EV-miRNAs, they strength the binding of miRNAs to cx43, suggesting that a multiprotein complex formed between RNA-binding proteins and Cx43 is important to select miRNAs.
NSMCE2 suppresses recombination and micronuclei formationScheme illustrating the Nsmce2 conditional allele (Nsmce2lox) used in this study.Figure 5Scheme illustrating the Nsmce2 conditional allele (Nsmce2lox) used in this study. A cross with Flp recombinase‐expressing mice eliminated the FRT‐PGK‐neo‐FRT cassette present on the initial allele (Nsmce2lox‐neo) (see ). In the conditional allele, exon 3 is flanked by loxP sites allowing for its deletion in the presence of Cre recombinase. NSMCE2 levels assessed by WB in UQ.CreERT2 transgenic Nsmce2+/+ and Nsmce2lox/lox littermate MEFs, 24 h after exposure to 4‐OHT. Tubulin levels are shown as a loading control. Clonogenic assay on immortalized UQ.CreERT2/Nsmce2lox/lox MEFs. The picture shows the colonies detected after 10 days of growth in the presence or absence of 4‐OHT, upon staining with crystal violet. NSMCE2 and BRCA1 foci in MMS‐treated (1 mM for 1 h; 24‐h recovery) UQ.CreERT2/Nsmce2lox/lox MEFs, 48 h after being treated (or not) with 4‐OHT. Scale bar, 5 μm. SCE events per metaphase on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h. ***P 0.001. Example of SCE events (arrows) found on Nsmce2‐deleted MEFs. SCE events at telomeres on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h, as detected by CO‐FISH (see ). Data are representative of three independent experiments. Error bars indicate SD. *P 0.05. Telomere lengths of UQ.CreERT2 Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h. Example of the intensity of telomere signals (yellow) used for (H). Percentage of cells with micronuclei found on cultures of UQ.CreERT2/Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.