EMBO/sd-character-level-ner huggingface dataset:
Figure 2Schematic strategy for generating PAX6 knockout in hESCs. The exon 8 is shared by all the isoforms and was replaced by the PGK-Puromycin element. The primers indicated in the panel are used to detect the gene knockout. qPCR quantification of PAX6 expression in Wildtype and PAX6 KO during retinal differentiation (n=3 biological replicates for each group). Western blot detection of PAX6 in Wildtype and PAX6 KO cells at day 20 of retinal differentiation. Immunostaining of PAX6 in Wildtype and PAX6 KO cells. C'-PAX6 antibody detects PAX6A, B and D. N'-PAX6 antibody detects PAX6 A and B but not D. Scale bars: 50 µm. qPCR quantification of retinal related genes in Wildtype and PAX6 KO cells. Samples are collected at day 6, 10, 14 and 19 after the start of differentiation.
Disease-causing mutations influence the association of CSPα with partner proteins.Figure 6A Overview of the targeted PPI screen. CSPα-mCit-PA was screened in two independent experiments against a focused library of 125 NL-tagged presynaptic proteins. Venn diagram depicting the 42 identified CSPα interaction partners. Domain structure of CSPα. The J-domain and the cysteine-string domain (CSD) are depicted. The ANCL-causing mutations ΔL116 and L115R are indicated. Tukey box plots of the mean cBRET (B) ratios obtained from PPI experiments with wild-type CSPα-mCit-PA and its mutant variants. Tukey box plots of the mean cLuC ratios (C) obtained from PPI experiments with wild-type CSPα-mCit-PA and its mutant variants. D Number of screened presynaptic proteins manually annotated according to their function and localization in the presynaptic terminal. Number of proteins identified with wild-type (wt) CSPα-mCit-PA or its mutant variants (ΔL116 and L115R) are indicated. E Influence of mutations in CSPα (ΔL116, L115R) on the interactions with ZDHHC17 in primary hippocampal neurons. BRET and LuC ratios were systematically quantified for interactions with wild-type and mutant CSPα fusion proteins. F Immunoblot analysis of SHEP cells transfected with plasmids encoding wild-type CSPα-mCit-PA and mutant variants. Blot was developed with an anti-CSP antibody; loading control: anti-Histone-H3 antibody. Arrowhead and bracket indicate monomeric CSPα-mCit-PA and SDS-insoluble protein aggregates, respectively. G Immunofluorescence image of SHEP cells co-transfected with NL-ZDHHC17 and CSPα-mCit-wt, -ΔL116 or -L115R constructs lacking the PA-tag. NL-ZDHHC17 was stained with an anti NanoLuc antibody. Scale bar: 20 µm.
Rescue of motor behavior in dCAM and AAV-dCAS animals 13 weeks after injection.Figure 7A, Gait analysis using the CatWalk XT system reveals motor defects in 6-OHDA lesion model. Both dCAM and AAV-dCAS animals transduce with AAV containing specific gRNAs show a significant improvement in different aspects of voluntary movement like the average speed of tread. Naive vs. 6-OHDA P=0.0063. AAV-dCAS: GFP vs. ALN P=0.015 multiple comparison ANOVA F(2,17)=12.81) Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition. the stride length of front paws: B, Naive vs. 6-OHDA P=0.0164. AAV-dCAS: GFP vs. ALN P=0.005, ALN vs. ALNe-218 P=0.0042, multiple comparison ANOVA F(4,22)=9.9) Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition. the duty cycle of left front paws. C, Naive vs. 6-OHDA P=0.0096. dCAM: GFP vs. ALN P=0.036 and ALN vs. ALNe-218 P=0.0252, multiple comparison ANOVA F(2,20)=5.199). Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition. D, In contrast to this, dopamine dependent drug-induced behavior does not show rescue effects: amphetamine-induced rotation analysis: change in rotational behavior in lesioned animals upon treatment with dopamine releaser substance. Net rotation = ipsilateral rotation-contralateral rotation (naïve vs. 6-OHDA P=0.09). Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition. CatWalk error bars represent mean ± SD. Rotation analysis error bars represent mean ± SEM.
Figure 2(A) MALT1 interacts with CARD14sh in a BCL10-dependent manner. HEK293T cells were transfected with FLAG-CARD14sh, Myc-MALT1 and FLAG-BCL10 as indicated. 24 h later, MALT1 was immunoprecipitated (IP) from cell lysates with anti-MALT1. CARD14sh and BCL10 co-immunoprecipitation with MALT1 was detected by immunoblotting with anti-FLAG. Immunoprecipitated MALT1 was detected by immunoblotting with anti-myc. The closed arrowhead shows the position of CARD14, the double arrowhead shows the position of BCL10. The asterisk indicates a non-specific band. Total expression levels of transfected proteins are shown by immunoblotting of a fraction of the cell lysates with the indicated antibodies (bottom panel). (B) MALT1 co-expression potentiates CARD14sh-induced JNK and p38 MAP kinase activation. HEK293T cells were transfected with the indicated concentrations of FLAG-tagged CARD14sh, with or without MALT1 (30ng). Transfection with FLAG-TRAF6 and FLAG-CARD11(L232LI) were used as a positive control. c-Jun accumulation, JNK and p38 phosphorylation were analyzed by immunoblotting with the antibodies indicated. Actin was used as a loading control. (C) MALT1 co-expression potentiates NF-kB activation by pathogenic CARD14 mutants. HEK293T were transfected with NF-kB reporter plasmid and wild-type (WT) FLAG-CARD14 or the indicated psoriasis-associated CARD14 mutants (15ng), with or without MALT1 (15ng). Luciferase activity in cell lysates was measured after 24 h. Values are the mean of triplicates ± s.e. Significance levels, *P<0.05, **P<0.01 and ***P<0.001 by student t test. Expression of transfected proteins was verified by western blotting (bottom panel). (D) CARD14(E138A) mutation enhances MALT1 binding. HEK293T cells were transfected with wild-type (WT) FLAG-CARD14sh or the indicated CARD14sh mutants and Myc-MALT1. 24 h later, MALT1 was immunoprecipitated (IP) from cell lysates with anti-MALT1 and CARD14 co-immunoprecipitation was detected by immunoblotting with anti-FLAG. Immunoprecipitated MALT1 was detected by immunoblotting with anti-MALT1. The asterisk indicates a non-specific band. Immunoprecipitation with a non-relevant antibody (rIgG) was used as a negative control (first lane). Total expression levels of transfected proteins are shown by immunoblotting of total cell lysates (bottom panel). Data are representative of two independent experiments.
CCR1 inhibition suppresses IMC recruitment and tumour development in vivo.Figure 4CCR1 immunofluorescence of primary IMCs (left, co-stained with Mac2) and AT2 cells (right, co-stained with E-cadherin) detected by CLSM. Scale bars, 10 μm. CCR1 staining was mainly detected in cytoplasmic vesicles within IMCs, but some CCR1 fluorescence was also detected at the plasma membrane along with Mac2 (inset in the left microphotograph). CCR1 immunohistochemistry of lung sections from BVE mice showing CCR1 expression in stroma IMCs. Scale bar, 50 μm. CT imaging of lungs of AdCre-infected Braf+/LSL−V600E mice treated with vehicle (top) or CCR1 inhibitor (bottom). H: heart; * indicates a tumour region accompanied by atelectasis. H&amp;amp;E staining of lung sections from vehicle (left) or CCR1 inhibitor (right) treated AdCre-infected Braf+/LSL−V600E mice. Scale bars, 250 μm (top) or 50 μm (bottom). Flow cytometry analysis of CD11c+ (top, green) and SPC+ (bottom, red) cells in the lung of AdCre-infected Braf+/LSL−V600Emice treated with vehicle (left) or CCR1 inhibitor (right).LungCD11c+ (left) and SPC+ (right) cell numbers (per left lobe) were quantitated in AdCre-infected Braf+/LSL−V600Emice treated with CCR1 inhibitor or vehicle (V) (n = 3, mean + SD).% LungCD4+ (left) and CD8+ (right) T lymphocytes were quantitated in AdCre-infected Braf+/LSL−V600E mice treated with CCR1 inhibitor or vehicle (V) (n = 3, mean + SD).
Figure 1A. Equal aliquots (2 mg) of a Triton X-100 solubilized lysate from rat brain were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins. B. Brain lysate as in (A) was incubated with GST or GST-nCLCb. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins. C. Full-length GST-nCLCb (1-228) and various truncation mutants were purified from bacteria. Equal aliquots of purified protein were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibody against CLCs. D. Brain lysate as in (A) was incubated with GST or GST-nCLCb proteins with boundaries as indicated. Specifically bound proteins were detected by Western blot with antibody against CLCs. E. Representation of a triskelia composed of three CHCs (orange) with three associated CLCs (blue). F. Highly enriched triskelia were stripped from purified CCVs. The triskelia were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins. For A-D/F, aliquots of the starting material (SM) equal to 10% of that added to the beads, was analyzed in parallel.
De novo transformation drives extensive redistribution of EZH2 A.Figure 1Schematic representation of the fibroblast-based model of cancer development used in this study. Red strikethrough represents inhibition of p53 and pRB by SV40 large and small T-antigens. Quantification by western-blot analysis of EZH2 and H3K27me3 levels at each stage of de novo transformation. UT: untransformed, PN: pre-neoplastic, TR: transformed. Histone H3 is used as a loading control. The graph on the right displays the H3 normalized densitometric values of western-blot band from untreated and DMSO treated cells. Values represent mean ± SEM from three biological replicates. One asterisk indicates p-value < 0.05 (one-tailed unpaired Student's t-test). ns: non-significant. Venn diagrams showing the overlap between H3K27me3-associated EZH2 binding sites detected at each stage of de novo transformation. Volcano plot showing the relative enrichment of EZH2 binding at all sites detected in either untransformed or transformed cells. Numbers within the volcano plot indicate differential binding sites with a p-value ≤1e-20 and a fold-change in normalised tag count ≥1.5. Enrichment significance calculated based on a Poisson distribution using the 'getDifferentialPeaks' function in homer (see: methods). P-values of > 1e-100 were set to 1e-100 for display reasons. Tracks on the right show representative high- and low-magnitude EZH2 differential peaks. ChIP-seq signal normalised to sequencing depth is shown. Tracks are scaled to be of the same height to make samples comparable. Blue bars represent regions called as an EZH2 binding site. UT: untransformed, TR: transformed. Neural-related GSEA gene signatures (left) enriched amongst genes with a high-magnitude, differential EZH2 binding site at their promoter (±5kb transcription start site (TSS)). Gene signatures are derived from GSEA Curated and Gene Ontology gene sets. The following gene signature names were modified from their original molecular signature database descriptor for clarity: 'CpG high promoters marked with H3K27me3 in the brain' - 'MEISSNER_BRAIN_HCP_WITH_H3K27ME3', 'CpG high promoters marked with H3K27me3 and H3K4me3 in the brain' - 'MEISSNER_BRAIN_HCP_WITH_H3K4ME3_AND_H3K27ME3', 'CpG high promoters marked with H3K27me3 in neural progenitor cells' - 'MIKKELSEN_NPC_HCP_WITH_H3K27ME3'. Functional classification of differential site-associated genes (right) (total genes: 37) that are found in the gene signatures 'Central Nervous System Development' and 'Neurogenesis'.
Figure 5(A) 1.0 x 106 bone marrow mononuclear cells (BM-MNCs) from controls (Npm1F/FVav1Cre-), Npm1 cKO (Npm1F/FVav1Cre+), p53 KO (Npm1F/Fp53-/-Vav1Cre-) and DKO (Npm1F/Fp53-/-Vav1Cre+) mice were transplanted into irradiated recipient mice with 4.0 x 105 competitor (C57/BL6 Ly5.1) BM-MNCs. (B) Survival curves of recipient mice are shown (n = 9-10 mice per group). Log-rank test was used to generate p values, **** p <0.0001. (C) Quantification of total BM-MNCs in Ctrl mice and lethargic/dead Npm1 cKO mice (n = 4 mice per group). (D) Representative flow cytometry analysis and quantification of KL Sca1-, KSL cells and HSCs (KSL CD150+CD48-) in lethargic/dead Npm1 cKO mice (n = 3 mice per group). (E and F) Percentages of CD11b+ and B220+ in PB (E), and c-Kit positivity in myeloid fraction in PB (F left) and BM (F right) of p53 KO and lethargic/dead DKO mice (n = 8-9 mice per group).
Figure 5(A) Viral DNA polymerase inhibitor phosphonoacetic acid (PAA, 300 μg/ml) treatment suppressed Gadd45b mRNA expression. Latently-infected SCG neurons were treated with LY294002 for 18 hrs to induce reactivation then treated with or without PAA until the termination time-points indicated. RNA was then collected and levels of Gadd45b mRNA quantified by RT-qPCR (n=3 biological replicates, the bars and error bars are mean ± SD). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. (B) Immunofluorescence images showing nuclear/cytoplasmic Gadd45b in latent neurons and cytoplasmic Gadd45b in viral DNA synthesis positive neurons. Latent SCG neurons treated with LY294002 and WAY-150138 for 72 hrs and then performed EdU pulse-labeling for detecting newly synthesized viral DNA followed by immunofluorescence staining probing with Gadd45b. The uninfected neurons served as control. Scale bar, 20 μm. (C) Immunofluorescence images showing nuclear-enriched Gadd45b in uninfected neurons at 72 hrs post treatment with LY294002. Scale bar, 20 μm. (D) The signal intensity quantification showing the ratio of nuclear/cytoplasmic Gadd45b at 72 hrs post treatment with LY294002 and WAY-150138 neurons. The signal intensity was quantified by ImageJ. 3 biological replicates, 10 EdU positive cells and 10 EdU negative cells were quantified for each replicate. The horizonal line and error bars are mean ± SD. (E) The percentage of Gadd45b nuclear puncta-localized neurons were quantified from latent neurons treated with LY294002 and WAY-150138 for 72 hrs. Each dot represents each biological replicate; 20 cells were quantified for each replicate. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. (F) Combined smFISH and immunofluorescence images showing Gadd45b nuclear puncta-presenting neurons were also LAT-positive. Reactivation was induced by treatment with LY294002 and WAY-150138 for 72 hrs. Scale bar, 20 μm. (G) Latent SCG neurons treated with LY294002 and WAY-150138 for 72 hrs, the Gadd45b nuclear puncta-presenting neurons were ICP4 negative (none observed in over 60 Gadd45b nuclear puncta-positive neurons), but a small population (~5%) in Gadd45b nuclear-enriched neurons were ICP4 positive. Scale bar, 20 μm.
Immunostaining of indicated differentiated iPSC cultures at DIV65.f2Immunostaining of indicated differentiated iPSC cultures at DIV65. Cells were stained for TH (red) and β-TubIII, FOXA2, NURR1, GIRK2 and VMAT2 (all green). Cell nuclei were counterstained with Hoechst (blue). (Bars, 20 μm).
Atg9- and WIPI1, but not DFCP1, are required for the generation of replicative BCVs.Fig 8HeLa cells were infected with B. abortus, and then transfected with each siRNA at 1 hr p.i. (A) Intracellular growth of B. abortus within control (blue bars), Atg9-knockdown (red bars), WIPI1-knockdown (green bars), and DFCP1-knockdown (purple bars) cells. CFUs were counted at the indicated time points after infection. Data are means ± SD from three independent experiments. **: p<0.01. (B, C) Representative confocal micrographs of control (top row), Atg9-knockdown (second row), WIPI1-knockdown (third row), and DFCP1-knockdown (bottom row) cells double-stained for Lamp2 (green) and B. abortus (red) at 24 hr p.i. BCVs co-localized with Lamp2 are indicated by arrowheads. The infected cells are outlined with white dashed lines. Scale bars are 10 μm. The percentage of Lamp2-positive BCVs was determined, and is shown in the line graph (C).
Quality marker panel for blood coagulation.Figure 3Preparation of plasma and serum samples. EDTA was used as anti-coagulation agent and incubation and centrifugation values are indicated. Volcano plot comparing 72 plasma vs. 72 serum proteomes. Proteins highlighted in yellow were chosen according to their p-value as markers for coagulation. Only the plasma enriched proteins (compared to serum) were used in the calculation of the coagulation contamination index. Ratio of the summed intensities of all plasma or serum proteins to the sum of the plasma-enriched panel proteins are plotted for all samples. Whiskers indicate the 10-90 percentile. Overlap of the three quality marker panels.
Oligo#5 influences the ratio of the two serotonin 2C receptor isoforms and changes the localization of the full-length receptor.Figure 4A. Structure of the reporter gene introduced into cells. The start codon in exon III is shown (circle) and the splicing patterns emerging from using the distal and proximal splice site (DS, PS). Skipping of exon Vb causes a frameshift, which terminates the protein in the first stop codon (square). Exon Vb inclusion keeps a longer open reading frame, that terminates after the GFP sequence. The encoded proteins are schematically shown below. B. Effect of oligo#5 on pre-mRNA splicing of the reporter gene, stably transfected into HeLa cells. C. Effect of oligo#5 on the truncated 5HT2C protein. Stable cell lines expressing the construct shown in panel (A) were treated with oligo#3 and oligo#5. After 48 hrs cells were separated in membrane containing fractions and soluble cytosolic supernatant. The fractions were analyzed by Western Blot using the anti-RNA1 antiserum (Figure EV1). The protein samples were on the same membrane, but the supernatant fractions were exposed about ten times longer.D. Effect of oligo#5 on the full-length 5HT2C protein. The protein was prepared as in panel (C) and analyzed using a polyclonal anti-GFP antiserum.E. Quantification of panels C and D from three independent experiments. The ratio of protein signal after oligo#3 and oligo#5 treatment is shown for RNA1 (p=0.002) and RNA2 (p=0.0003), n=3, respectively. F, G. Effect of oligo#5 on localization of the EGFP-tagged serotonin 2C receptor. Confocal microscopy images of three representative pictures are shown.H. Quantitification of GFP signal found on the membrane after oligo#5 addition. The signal around the peripheral region was determined by visual inspection and calculated as a fraction of total signal using NIH image. 200 cells from three individual experiments were analyzed, p<0.008, n=200.
Model of aluRNA‐driven maintenance of the nucleolusThe knockdown of aluRNA or treatment with α‐amanitin induces the segregation of nucleolar compartments into smaller domains (dispersion process) as observed (Figs A and A, and Appendix ).Figure 9Model of aluRNA‐driven maintenance of the nucleolusThe knockdown of aluRNA or treatment with α‐amanitin induces the segregation of nucleolar compartments into smaller domains (dispersion process) as observed (Figs A and A, and Appendix Fig S2). With regard to rRNA production, those "droplets," which may vary in composition, are less efficient but some rRNAs are still produced albeit at low levels (Appendix Fig S1B and C and Fig C and D). Overexpression of aluRNA promotes assembly into larger nucleolar subcompartments, with enhanced Pol I activity (see Fig ). This model is in excellent agreement with the previously reported liquid‐like model of the nucleolus (Brangwynne et al, ; Weber Brangwynne, ) and suggests a RNA binding‐driven liquid‐like transition between dispersed nucleolar domains and intact and fully functional nucleoli.
Figure 1A-C ARR1/10/12 protein levels were examined by western blot when five-day-old seedlings of 35S:ARR1:MYC, 35S:ARR10:YFP, and 35S:ARR12:YFP were treated with or without 200 mM NaCl for the indicated time points. Actin was used as the internal reference. The relative intensity of band detected by anti-MYC or anti-GFP antibody to that by anti-Actin antibody without treatment was set to 1.0. D-F Quantification analysis of the protein levels of ARR1/10/12 in (A-C).
Figure 5CD31 and CD34 double positive endothelial cells were isolated from wild-type or EVL-/- P5 retinas by FACS and analyzed by RNA-sequencing. Two independent experiments; in total 18 retinas from WT mice and 18 retinas from EVL-/- mice from three different litters, each.(B) Pathway analysis (metascape.org) was used to identify differentially expressed gene sets in EVL-/- retinal endothelial cells. Selected gene sets involved in vessel branching are shown. (C) Pathway analysis of all under-represented gene sets in EVL-/- endothelial cells. (D) Analysis of mRNA levels of Esm1, Ptgs2 (cyclooxygenase 2), serpine 1 and paxillin in EVL-deficient MLEC relative to WT controls set to 100. n = 3-4 independent experiments, error bars represent SEM, one sample t-test, *p<0.05; **p<0.01. (E) ESM1 protein expression in P5 retinas of wild-type (WT) and global EVL-/- mice. Retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and antibodies directed against the tip cell marker ESM1 (red). Representative images from three independent experiments are shown. Scale bars, 100 µm. (F) Analysis of retinal ESM1 protein levels normalized to wild-type littermates. Error bars represent SEM; ***p<0.001, unpaired Student's t-test; three different litters.