EMBO/sd-character-level-ner huggingface dataset:
Figure 6Depletion of piRNAs from chr7 piRNA cluster RMER4B sequence in chr7 piRNA clusterΔRMER4B/ΔRMER4B mice. Top panel shows overall view of chr7 piRNA cluster, and lower panel shows magnified view of RMER4B region in chr7 piRNA cluster. Only uniquely mapped piRNAs are shown. Depletion of piRNAs that potentially target pit-RNA in chr7 piRNA clusterΔRMER4B/ΔRMER4B mice. piRNAs are mapped to RMER4B region of Rasgrf1 DMR (deleted sequence in Rasgrf1ΔRMER4B/ΔRMER4B mice) allowing two mismatches. Black bars represent piRNAs that are mapped to this region in antisense direction to pit-RNA. The numbers of genomic loci where each of these piRNAs is perfectly matched (loci), their read numbers (reads) and mismatches in the alignment between these piRNAs and Rasgrf1 RMER4B sequences (mm). All piRNAs shown here were perfectly matched to chr7 piRNA cluster. The numbers in parentheses show the total number of small RNA reads mapped to the genome. DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatogonia from chr7 piRNA clusterΔRMER4B/ΔRMER4B mice. DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatocytes, spermatids, and sperms (D) from chr7 piRNA clusterΔRMER4B/ΔRMER4B mice. DNA methylation pattern in the progeny of male Rasgrf1ΔRMER4B/ΔRMER4B mice. DNA methylation status of the Rasgrf1 DMR in E12.5 whole embryos is shown. E12.5 whole embryos were obtained by the crosses shown in the panel. Two independent bisulfite-treated DNA sample were examined for Homo13 embryo.
Figure 2A. Representative H&amp;amp;E staining of whole lungs from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. Scale bar: 5 mm. B. Quantification of the tumor/lung ratio. n=7 for each group except for EGFRL858R/Rhob+/- placebo (n=6). C. Representative Ki67 immunostaining of EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D). Three independent zones/mice lung were used for quantification. n=21 (7 mice) for each group except for EGFRL858R/Rhob+/- placebo (n=18; 6 mice). E. Immunostaining of cleaved caspase-3 in lung tumors from EGFRL858R/Rhob+/+ or EGFRL858R/Rhob-/- mice treated for 24 h with erlotinib at 12.5 mg/kg. Scale bar: 50 µm. (**: p<0.001 vs. placebo; ***: p<0.0001 vs. placebo).
Figure 2PRMT1 interacts with METTL14 in cells. HEK293 cells expressing GFP-tagged PRMT1, PRMT3, or PRMT6 were lysed and immunoprecipitated with an anti-METTL14 antibody, followed by Western blot analysis using an anti-GFP antibody. * indicates the location of the IgG heavy chain. Endogenous METTL14 interacts with PRMT1. The reciprocal endogenous co-immunoprecipitation (IP) assays were performed using the METTL14 antibody for IP and the PRMT1 antibody for Western blot detection (left panel) and using the PRMT1 antibody for IP and the METTL14 antibody for Western blot detection (right panel). The C-terminal IDR of METTL14 is essential for its interaction with PRMT1. HEK293 cells were transfected with GFP-tagged PRMT1 and Flag-tagged FL or C-terminal IDR-truncated (1-400) METTL14. IP was performed using an anti-GFP antibody, and Western blot analysis was performed using anti-GFP and anti-Flag antibodies. GST pull-down detection of the interactions of PRMT1 with GST-tagged FL and truncated (1-400) recombinant METTL14. The black triangles indicate recombinant METTL14 proteins. Overexpression of PRMT1 enhances METTL14 arginine methylation. HEK293 cells were transfected with either GFP vector or GFP-PRMT1, together with Flag-METTL14. The level of METTL14 methylation was detected by IP using an anti-Flag antibody, followed by Western blot analysis using an anti-ASYM26 antibody. * indicates the location of the IgG heavy chain. Knockdown of PRMT1 expression reduces METTL14 arginine methylation. HEK293 cells were transfected with control siRNA (siCtrl) and the siRNA targeting PRMT1 (siPRMT1). METTL14 was immunoprecipitated from these cells, and its methylation level was detected by Western blot analysis using an anti-ASYM26 antibody. * indicates the location of the IgG heavy chain.
Figure 5A, Co-IP analysis was performed in 293T cells with antibodies against p53 or H2Bub1. B, peptide sequence synthesized according a previous report and used for peptide pull-down assay. Peptide pull-down assay was performed with synthesized peptides D, Co-IP analysis for H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) treated with a USP7-specific inhibitor P5091. E, A proposed working model of the regulation of H2Bub1 by p53.
Figure 4A Detection and conformation of O-GlcNAc modification of His-ATX1ΔN catalyzed by His-SECΔN in vitro. His-ATX1ΔN (expressing residues 592-952 of the C-terminus) and His-SECΔN (expressing residues 801-1062 of the C-terminus) were recombinantly expressed and affinity purified separately. O-GlcNAcylation of His-ATX1ΔN was detected by anti-CTD110.6 antibody, and further conformed by β-elimination analysis. B His-SECΔN activates His-ATX1ΔN in vitro. Histone methyltransferase activity of ATX1ΔN was detected with or without recombinant SECΔN. H3K4A: mutated H3 in which the fourth amino acid, a lysine (K), was replaced with alanine (A). C Analysis of ATX1 O-GlcNAc modification in wild-type and sec-5 plants. Total soluble protein extracts from 12-d-old seedlings were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. CBB: Coomassie brilliant blue staining, showing relative protein loading amount. D Loss of SEC function reduced ATX1 activity in Arabidopsis. Nuclear proteins were extracted and ATX1 was immunoprecipitated with anti-ATX1 antibody from wild-type and sec-5 mutant plants, respectively, and then used for histone H3K4 methyltransferase activity analysis with recombinant H3 as catalyzing substrate. Band intensities were quantified with Image J. The H3 signal was first normalized by input signal and then was used for H3K4me3 signal normalization. E Comparison of ATX1 protein levels in Col-0 and sec-5 plants at stages before and after flowering. Seedlings were collected for protein extraction at 21 days (before flowering) and 35 days (after flowering) after seeds were planted on plates. Anti-ATX1 antibody was used for immunoblot assay.
Figure 2(A) Schematic showing the possible replication products and oligonucleotide restart products for the assay in (B). Green lines represent the restart oligonucleotides and the dashed line indicates extension of the stall product which will occur due to bypass of the Tg after oligonucleotide binding. (B) Oligonucleotide competition assay on UD and Tg templates. S; scrambled oligonucleotide.(D) Pulse chase reaction on UD and Tg templates performed as shown. Where present, Pol δcat (10 nM) was added directly after addition of the chase. (E) Standard replication reaction performed in the absence and presence of RFC/PCNA on the Tg template.
Figure 3A) Schematic representation of the in vitro scratch cell migration/ wound healing assay. B) Icos1 at 18 nM F-domain stimulates cell migration relative to PBS/vehicle control. Scale bars are 100 µm. C) Comparison of cell migration induced by Ang1, Ang2, and designed F-domain scaffolds after 12 hours of treatment. The change in wound area is normalized to vehicle control. All experiments have at least 3 biological replicates. (*) is the comparison between F-domain scaffolds vs Ang1, and (#) is the comparison between F-domain scaffolds vs Vehicle. 0.0001.D) Schematic of in vitro tube formation assay; F-domain scaffolds (designed scaffolds) were added at 90 nM F-domain and removed at 24h; vascular stability was analyzed at 48h and 72h timepoints. E) Representative increase in vascular stability by Icos1. The number of nodes (blue), meshes (red), and tubes (green) were quantified using angiogenesis analyzer plugin in ImageJ. Scale bar is100 µm. F) Effect of F-domain scaffolds on the vascular stability at 48- and 72-hour timepoint were assessed by taking the average of the number of nodes, meshes, and tubes and normalized to vehicle as log2 fold change. All experiments have at least three biological replicates except for Ang2, Tet1, Tet2, akC4, and Icos2 at 72-hour timepoint in tube formation assay that have two biological replicates. G) Designed F-domain scaffolds fall into Ang1- and Ang2-like classes. Color indicates highest (1) to lowest (0) pAKT, pERK1/2, and pFAK level which were column normalized. Cell migration level and vascular stability (48-hour timepoint) were also column normalized from highest to lowest.
Figure 8A - Schema of experimental design. At 40 days in culture, human cerebral organoids were electroporated with one of the indicated DNA plasmid combinations, followed by 7 additional days in culture prior to analysis. B-G - Stains and quantifications of human cerebral organoids electroporated with the indicated construct combinations, to reveal the frequency of electroporated cells remaining as progenitors (%GFP+Ki67+/GFP+). Histograms represent mean ± SEM; symbols in plots indicate values for individual organoids; X2-test, *P < 0.05. N = 8-11 replicates per group. Dashed lines indicate ventricular surface, solid arrowheads indicate Ki67+/GFP+ cells, open arrowheads indicate Ki67-/GFP+ cells. Scale bars, 50 µm. H - Schematic drawing of conclusions from this study. Let-7 expressed in the early telencephalic neuroepithelium represses the expression of p53 and Irs2, which maintains the cellular homeostasis and epithelial integrity. Loss of let-7, as in Dicer1 mutants, de-represses expression of Irs2 and of p53 pathway components, which further increase Irs2 levels. High Irs2 enhances progenitor cell proliferation and loss of apical adhesion, altering neuroepithelial homeostasis and integrity, and leading to the formation of hyperproliferative rosettes.
Figure 4A Quantification of peripheral ER structures in WT and ∆ice2 cells (SSY1405, 1603) treated with 8 mM DTT for the times indicated. Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; n.s., not significant. B Flow cytometric measurements of GFP levels of WT and Δice2 cells containing the transcriptional UPR reporter (SSY2306, 2312). Cells were treated with 8 mM DTT for the times indicated. Data were normalized to untreated WT cells. Mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant. C, D Fluorescence images of cortical sections of WT and Δice2 cells expressing Sec63-mNeon and Rtn1-mCherry (SSY1405, 1603) that were untreated (C) or treated with 8 mM DTT for 1 h (D). E Quantification of WT and ∆ice2 cells with Rtn1-mCherry puncta after treatment with 8 mM DTT for the times indicated. Mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; n.s., not significant. F Quantification of peripheral ER structures in untreated WT and UPR-deficient ∆hac1 cells (SSY2228, 2331), overexpressing ICE2 from plasmid pSS761 where indicated. Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01. G Flow cytometric measurements of GFP levels of WT and Δhac1 cells containing the UPR reporter (SSY2306, 2314) and overexpressing ICE2 from plasmid pSS761 where indicated. Data were normalized to untreated WT cells. Mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with the corresponding untreated sample, as judged by a two-tailed Student's t-test assuming equal variance. Exception was the test against the normalized value for wild-type cells, for which a two-tailed Student's t-test with unequal variance was applied. n.s., not significant..
Figure 2(A) Immunofluorescence (IF) staining showing Siglec-F and Iba1 localization in hippocampus CA3 region of 3wk induced CK (top) or CK-p25 (bottom) mice. Scale bars = 50 μm. Colors are: Magenta = Iba1, Cyan = Siglec-F. Images are 60x super-resolution max z-stack projections taken from coronal slices. (B-C) Percent area coverage of (B) Iba1 and (C) Iba1-proximal Siglec-F between CK and CK-p25 mice across hippocampal regions. Bars indicate mean ± 95% CI; n = 3-4 animals; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. (D-E) IF staining of Siglec-F and Iba1 in (D) 5XFAD 2 month hypothalamus (HY) and (E) 5XFAD 6 month dentate gyrus (DG). Yellow = Aβ from methoxy X04 (D) or D54D2 (E). Scale bars = 50 μm. (F) Percent area coverage of Iba1-proximal Siglec-F relative to total Iba1 across all analyzed thalamic, cortical, and hippocampal regions of 6 month WT (non-Tg) and 5XFAD mice. Box indicates quartiles and whiskers indicate the last datum within 1.5 inter-quartile range; n = 15-40 images (3-5 animals); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.(H) Cumulative distribution function (CDF) of the distance between Siglec-F+ and Iba1+ mask voxels and nearest Aβ plaques in 6 month 5XFAD mice. Each value in the CDF represents the mean distance calculated from a single z-stack of a ROI containing ≥ 1 plaque of size ≥10 μm2. n = 55 images (4 animals). (I-J) Relative RNA abundances for Siglec-8 in (H) hippocampus and (I) posterior cingulate cortex post-mortem patient tissue. Linear regression R2 values are shown on plots. (K) IF staining showing Siglec-8 and Iba1, and MHC-II in cortical white matter tissue from a patient with late-onset AD (20x wide-field; Case ID: 01-43). Scale bars = 50 μm. Images are 60x super-resolution max z-stack projections. (L-M) Percent area coverage of Siglec-8 localized to Iba1 or MHC in individuals with non-AD or AD. In (K) each point represents one WM or GM region average from each individual. In (L) values and statistics are shown for each image analyzed. Boolean mask operators indicate: ° = mask dilation, ⋂ = intersection, ⋃ = union, NOT = inverse mask. Box indicates quartiles and whiskers indicate the last datum within 1.5 inter-quartile range; n = 8-43 images (n = 1 ND, 3 early-onset AD, 4 late-onset AD); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.
KSHV‐TK associates with the p85 regulatory subunit of PI3‐KinaseA schematic representation of the p85 regulatory subunit SH2 consensus binding motif of PI3‐Kinase in KSHV‐TK.Figure 8A A schematic representation of the p85 regulatory subunit SH2 consensus binding motif of PI3‐Kinase in KSHV‐TK. B Immunoblot analysis reveals that GFP‐KSHV‐TK WT and Y2F but not Y120F associate with p85 of PI3‐Kinase. C Immunoblot analysis of p85PI3‐Kinase antibody immunoprecipitates reveals that GFP‐KSHV‐TK and its Y2F mutant but not Y120F associate with p85 of PI3‐Kinase. D Immunoblot analysis of CrkII and CrkL antibody immunoprecipitates reveals that GFP‐KSHV‐TK and the p85PI3‐Kinase associate with CrkII and CrkL. No association is observed with the Y2F, Y3F or kinase‐dead TK. E Tyrosine phosphorylation of CrkII and CrkL is only upregulated in cells expressing GFP‐KSHV‐TK. F Immunoblot analysis reveals that recombinant His‐CrkII binds to and is phosphorylated by KSHV‐TK but not the Y2F or Y3F mutants.
Figure 1(A RNA sequencing of CD31 and CD34 double-positive retinal endothelial cells from P5 wild-type mice. (A) RNA levels (FPKM; fragments per kilobase million) of marker genes of endothelial cells (green; CD31 (gene name PECAM1), CD34, von Willebrand Factor (vWF), tyrosine-protein kinase receptor Tie2 (TEK), VE-cadherin (CDH5), endoglin (ENG), CD146 (MCAM) and VEGFR2 (KDR)), astrocytes (blue, GFAP), immune cells (red; T-cells (CD3E), B-cells (CD19), all leucocytes (CD45, PTPRC), and monocytes/macrophages (F4/80, EMR1)), Müller glia cells (orange; aquaporin 4 (AQP4)), neurons (yellow; retinal ganglion cells (RNA binding fox-1 homolog 3, RBFOX3), amacrine cells (parvalbumin, PVALB), bipolar cells (PKC-α, PRKCA), horizontal cell (calbindin, CALB1), photoreceptors (rods, CD73 (NT5E); cones, transducing (GNAT1)), and retinal pigment epithelial cells (magenta; retinal pigment epithelium-specific 65 kDa protein (RPE65). B) RNA sequencing of CD31 and CD34 double-positive retinal endothelial cells from P5 wild-type mice. (B) RNA levels of VASP (red), EVL (green) and Mena (blue) in the CD31 and CD34 double-positive P5 retinal endothelial cells. Error bars represent SEM; n = 18 animals (36 retinas) from six independent litters; four independent experiments. ***p<0.001, one-way ANOVA with Bonferroni's multi comparison test. (C-E) Staining of Ena/VASP proteins in blood vessels of P5 mouse retinas. P5 wild-type mouse retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and VASP
Reducing mitochondrial ROS levels in ADAM17-/- mutants leads to a rescue of the LD accumulation and retinal degeneration.Figure 5A-D. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (A, B) wild type; (C, D) ADAM17-/- mutants, reared either on DMSO or AD4. Scale bars: 10μm. E. Integrated total lipid counts from retinas corresponding to the genotypes and treatments mentioned above; n=10 for each. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001. F-I. TEM images of 2 week aged retinas corresponding to either wildtype or ADAM17-/- mutants grown on either DMSO or AD4. Scale bars: 10μm. J. Percentage of normal, abnormal or missing rhabdomeres from retinas corresponding to the genotypes and treatments mentioned above. n=180 ommatidia from 3 different fly retinas for each. K-N. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (K) ADAM17-/- ; (L) glial specific SOD2 over-expression in an ADAM17-/- mutant; (M) glial specific SOD1 over-expression in an ADAM17-/- mutant; (N) neuronal specific SOD2 overexpression in ADAM17-/- mutant. Scale bars: 10μm. O. Quantitation of the BODIPY signal shown as Integrated total lipid for the genotypes mentioned above, n=10 for each. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean, and the diamond an outlier. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001, **p<0.01, *p<0.05. P-Q. TEM analysis of 5 week aged retinas corresponding to either ADAM17-/- mutants or SOD2 over-expression in an ADAM17-/- mutant. Scale bars: 10μm. R. Percentage of normal, abnormal and missing rhabdomeres in ADAM17-/- mutant retinas, as compared to retinas overexpressing glial specific SOD2 in an ADAM17-/- mutant background observed with TEM; n=180 ommatidia from 3 different fly retinas for each. S. Oxyblot analysis of oxidative modification of proteins from retina lysates of wild type and ADAM17-/-. T. Analysis of Oxyblot intensity normalised to its respective actin controls for each genotype. n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01. Scale bars: 10μm.
Figure 5ChIP analysis of THP1-derived macrophages demonstrates APEX1 recruitment to the MARCKS promoter. RT-qPCR analysis of fragments of the MARCKS promoter region (a and b) or a non-specific region (nc) co-immunoprecipitated with anti-APEX1 antibody. ChIP analysis of APEX1 recruitment to the MARCKS promoter was performed except the cells expressed control shRNA (NC) or ROCKI shRNA and treated with Pam3 for 8 h. The y-axis shows the percentage of enrichment in normalized to input. An unrelated region of the ACTB2 promoter was amplified as a control. Left: Western blot analysis of APEX1 and HDAC1 in control rabbit IgG or anti-APEX1 antibody immunoprecipitates from NC (control) or ROCKI knockdown THP1-derived macrophages. Right: RT-qPCR analysis of ROCKI in the same immunoprecipitates. Knockdown of ROCKI decreased HDAC1 enrichment on the MARCKS promoter. ChIP-qPCR analysis of HDAC1 was performed using an anti- HDAC1 antibody. Knockdown of ROCKI enhances H3K27ac deposition on the MARCKS promoter. ChIP-qPCR analysis of H3K27ac was performed using an anti-H3K27ac antibody.
a, MEFs of RagA+/+, RagAGTP/+ and RagAGTP/GTP genotypes were deprived of amino acids for 1 h and re-stimulated for 10 min.f1a, MEFs of RagA+/+, RagAGTP/+ and RagAGTP/GTP genotypes were deprived of amino acids for 1 h and re-stimulated for 10 min. Whole-cell protein lysates were immunoblotted for indicated proteins. b, Total RNA was extracted from RagA+/+ (n = 3), RagAGTP/+ (n = 3) and RagAGTP/GTP (n = 2) MEFs and RagA mRNA expression determined by quantitative PCR (mean ± s.e.m.). c, Weights of RagA+/+ (n = 24), RagAGTP/+ (n = 52) and RagAGTP/GTP (n = 22) mice at birth (data are scatter dots, mean). d, Representative photographs of RagA+/+, RagAGTP/+ and RagAGTP/GTP neonates. Scale bar, 1 cm. e, Early suppression of mTORC1 signalling after birth was determined by immunoblotting of protein extracts from liver and heart of RagA+/+ (+/+), RagAGTP/+ (G/+) and RagAGTP/GTP (G/G) neonates immediately after Caesarean section (0 h) or after 1 h of fasting (1 h fast). f, Liver and heart extracts from RagA+/+, RagAGTP/+ and RagAGTP/GTP neonates fasted for 10 h were analysed by immunoblotting for the indicated proteins. g, Survival curve of fasted neonates. Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored (+/+: n = 13; G/+: n = 26; G/G: n = 10). h, Survival curve of fasted neonates treated with rapamycin. Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored (+/+: n = 13; G/+: n = 13; G/G: n = 6). Numbers indicate the median survival for each curve. *P<0.05; **P<0.01; ***P<0.005.