EMBO/sd-character-level-ner huggingface dataset:
The AAV-split-dCas9 Activator System (AAV-dCAS).Figure 3A, dCas9 is separated into a N- and a C-terminal part (AAV-N-dCas9aa1-573-N-intein and AAV-C-dCas9aa574-1368-VP64-C-intein), both portions are fused to the corresponding intein-moieties. Upon co-expression intein-mediated trans-splicing leads to reconstitution of Cas9 protein. Data information: Abbreviations: dN-Cas9 - N-terminal dCas9-residues 1-573, N-intein - N-terminal part of DNA polymerase III subunit alpha, dC-Cas9 - C-terminal dCas9 residues 574-1368, C-intein - C-terminal part of DNA polymerase III subunit alpha VP64 - 4x VP16 herpes simplex virus protein vmw65, TSS - transcriptional start site B, RT-qPCR analysis of Ascl1 induction comparing the activation capacity of full-length versus split-dCas9 in Neuro2A cells (data in fold change normalized to non-activated control; dCas9 6116 ± 847.3, split-dCas9 4415 ± 748.8, n=3 biological replicates, box-and-whiskers plots indicate median, 25th-75th percentile and min/max whiskers). C, Immunocytochemistry analysis of reprogrammed primary astrocytes cells 16 days after lentiviral transduction revealed successful in vitro reprogramming into neurons using CRISPRa. Astrocytes are infected with two lentiviruses expressing dCas9-VPR in a intein-split version similar to the AAV-dCAS system but driven by a Tet-O promoter. Tet-O driven dsRed and Ascl1 cDNA expressing construct were used as negative and positive controls respectively. Arrows indicate single MAP2 positive background neurons, arrowheads indicate double positive induced neurons. Scale bar indicates 20 µm. Data information: Abbreviations: OE - overexpression D, Schematic representation of the AAV-dCAS system: For induction of up to five endogenous genes plus a GFP reporter, a total of four different AAVs are utilized. dCas9 is delivered by two AAVs, a third AAV is needed for the delivery of the SAM activator. A fourth virus contains the reporter gene, while sgRNAs are distributed between the vectors. To ensure cell type specificity upon Cre expression, the N-dCas9 and the GFP are inverted and flanked by two different LoxP sites (LoxX and Lox511). Data information: Abbreviations: dN-Cas9 - N-terminal dCas9-residues 1-573, N-intein - N-terminal part of DNA polymerase III subunit alpha, dC-Cas9 - C-terminal dCas9 residues 574-1368, C-intein - C-terminal part of DNA polymerase III subunit alpha, VP64 - 4x VP16 herpes simplex virus protein vmw65, p65 - p65 subunit of human NF-ĸB, HSF1 - heat shock factor 1, MS2 - MS2 bacteriophage coat protein, PAM - protospacer adjacent motif, TSS - transcriptional start site, CBh - chicken β-actin hybrid promoter. SgRNA expression is driven by the different Pol III promoters (H1, hU6, mU6 and 7SK). E, Multiplexed activation of Ascl1/Lmx1a/NeuroD1, Ascl1/Lmx1a/Nr4a2 and Ascl1/ Lmx1a/ Nr4a2/Pitx3/FoxA2 in primary astrocytic cells. n=2-3 biological replicates, one representative run is shown, additional data in supplement. Activation levels are depicted as fold change between cells transfected with and without sgRNAs. All levels were normalized to β-Actin. Error bars represent mean ± SD between technical replicates.
Characterization of HCT116 and its tri‐ and tetrasomic derivatives.Figure 1(A) Chromosome paints of used tri‐ and tetrasomic cell lines. Bar-10 μm. (B) Growth curves of tri‐ and tetrasomic cell lines in comparison with their diploid counterparts. Each point represents the mean with standard deviation of three independent experiments. (C) Cell‐cycle progression of HCT116 (left panel) and HCT116 5/4 (right panel) after release from thymidine block, analyzed by flow cytometry. The major delay occurs in the G1 and the S phase. The length of each cell‐cycle phase is indicated above the graph. Population was considered to enter a specific phase of cell cycle if at least 50% of cells showed corresponding DNA content. See also Supplementary Figure S1. Source data is available for this figure in the Supplementary Information.
Figure 3(A) Surface expression of PD-1 in vivo in 99LN-BrM quantified by flow cytometry (n=3). (B) PD-L1 in 99LN BrM quantified by flow cytometry (n=3). (C) Flow cytometric analysis of PD-L1 in tumor-free brain and 99LN-BrM (n=3). (D) Flow cytometric analysis of PD-L1 expression on 99LN-BrM associated my myeloid cell types. (n=6). (E) Expression of genes associated with antigen presentation and co-regulatory factors in TAM-MG and TAM-MDM. n=3 per condition. Values are depicted as normalized counts. Genes labeled with # can have stimulatory effects depending on the co-regulatory receptor on T cells. (F) Gene expression of co-regulatory receptors in TIL-CD4 and TIL-CD8. n=3 per condition. Values are depicted as normalized counts. (G) Functional gene annotation of altered cellular pathways in TAM-MG vs. TAM-MDM. Left panel depicts pathways enriched in TAM-MG, right panel depicts pathways enriched in TAM-MDM. Adjusted p value (padj) were obtained by Wald test and corrected for multiple testing using the Benjamini and Hochberg method. (H) Representative HALO spatial plot showing the spatial distribution of cell types with indicated marker combination in 99LN-BrM and representative IF images of multiplexed histology. Scale bar; 100 μm. (I) HALO proximity histogram depicts the distance (μm) between the indicated cell types for individual mice (n=3).
Figure 6A AtANN1 current recordings in Xenopus oocytes. Whole-cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1, OST1, or AtANN1 + OST1 cRNA. Bath solution was described in Materials and Methods. The voltage protocol as well as time and current scale bars for the recordings are shown. B Current-voltage (I-V) relationship of the steady-state whole-cell currents in Xenopus oocytes described in (A). Negative current is influx of cations into the oocytes. C Percentage of total Xenophus oocytes in different nA range according to (A). D AtANN1 current recordings in oocytes. Whole-cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1, AtANN1S289A, OST1, AtANN1 + OST1, and AtANN1S289A + OST1 cRNA. E Current-voltage (I-V) relationship of the steady-state whole-cell currents in Xenopus oocytes described in (D). F Percentage of total Xenophus oocytes in different nA range according to (D).
Figure 1A Schematic of the control of lipid synthesis by estradiol-inducible expression of ino2*. B Sec63-mNeon images of mid and cortical sections of cells harboring the estradiol-inducible system (SSY1405). Cells were untreated or treated with 800 nM estradiol for 6 h. C Flow cytometric measurements of GFP levels in cells containing the transcriptional UPR reporter. WT cells containing the UPR reporter (SSY2306), cells additionally harboring an estradiol-inducible GAL promoter (SSY2307) and cells additionally harboring the system for estradiol-inducible expression of ino2* under the GAL promoter (SSY2308) were untreated, treated with 800 nM estradiol for 6 h, or treated with 8 mM DTT for 1 h. Data were normalized to untreated WT cells. Bars represent the mean of three biological replicates (n = 3), error bars are the standard error of the mean (s.e.m.). Asterisks indicate statistical significance compared with the corresponding untreated sample, as judged by a two-tailed Student's t-test assuming equal variance. Exceptions were the tests against the normalized value for wild-type cells, for which a two-tailed Student's t-test with unequal variance was applied. *, P < 0.05; n.s., not significant. WT, wild-type. D Western blot of Sec63, mCherry and Pgk1 from WT cells (SSY1404), cells harboring the estradiol-inducible system (SSY1405) or ∆opi1 cells (SSY1607), all of which expressed Sec63-mNeon and Rtn1-mCherry. Cells were untreated or treated with 800 nM estradiol for 6 h. Pgk1 served as a loading control. E Quantification of ER size in estradiol-treated cells harboring the inducible system (SSY1405), untreated ∆opi1 cells (SSY1607) and WT cells (SSY1404) treated with 8 mM DTT for 1 h. Bars represent mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with 0 nM estradiol or ∆opi1 cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; n.s., not significant.
Figure 8HUVECs transfected with control or CEP41 siRNAs were cultivated under hypoxia and their whole cell lysates were used for immunoblot assays of HIF1α. The HIF1α expression of hypoxic cells was compared to that of normoxic cells and the protein levels were normalized against β-ACTIN in the same blot. Co-immunoprecipitation assays using HIF1α-, and CEP41-specific antibodies were performed in HEK 293T cells expressing GFP-CEP41 under normoxia or hypoxia. An IP using IgG-specific antibodies was performed as a negative control. NOR, normoxia; HYP, hypoxia. The CEP41-depleted HUVECs were transfected with expression vectors encoding nothing (MOCK), CEP41, or HIF1α and cultivated under hypoxia. Whole cell lysates were used for immunoblot assays for phospho-AURKA and AURKA, and the resulting data were compared to those of normoxic and hypoxic control cells. Protein levels were normalized against β-ACTIN in the same blots. NOR, normoxia; HYP, hypoxia. The CEP41-deficient HUVECs were transfected with expression vectors encoding nothing (MOCK), CEP41, HIF1α, or HIF1α with CEP41 and exposed to hypoxia. VEGFA (E) and VEGFR2 (F) mRNA levels were analyzed by qRT-PCR using cDNA from transfected cells. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined using the one-way ANOVA followed by Tukey's post hoc test (***P < 0.001, ns: non-significant). NOR, normoxia; HYP, hypoxia. Control or CEP41-depleted HUVECs were transfected with expression vectors encoding nothing (MOCK) or HIF1α and treated with MG132 or not, and then the cells were cultivated under hypoxia. Whole cell lysates were used for immunoblot assays for HIF1α, phospho-AURKA, and AURKA, and the protein levels were normalized against β-ACTIN in the same blots. NOR, normoxia; HYP, hypoxia. Quantification of tube node number in (H) and tube length in (I) from in vitro angiogenesis assays with HIF1α #1 or #2 siRNAs-transfected HUVECs overexpressing nothing (MOCK) or CEP41 under hypoxia. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition. VEGFA (J) and VEGFR2 (K) mRNA levels were analyzed by qRT-PCR using cDNA from hypoxic HIF1α-depleted HUVECs transfected with control or CEP41 expression vectors. The results were compared to those of control and normoxic HIF1α-depleted cells. Data are shown as mean ± SD of three independent experiments. A schematic diagram showing the molecular mechanism by which CEP41 drives hypoxia-induced angiogenesis.
Figure 7a. A flow-chart showing the sample preparation for the SILAC-based relative quantification of K48/K63-branched ubiquitin chains from yeast by mass spectrometry. 10xHis-Ub(R54A) was overexpressed in wildtype cells (wt) and either in cells deleted for UBC1 (Δubc1) or cells expressing ubiquitin-binding-deficient Ubc1-EA. The R54A substitution in ubiquitin enables the production of a marker peptide for K48/K63-branched ubiquitin by trypsin digest, which can be detected by targeted proteomics (PRM). B. Log2 transformed fold changes of peptides between the Δubc1 strain and wildtype were determined. The forward and reverse SILAC experiments are shown on the x or y axis, respectively. The different ubiquitin marker peptides are indicated in the figure. C. Experiment as in (B) but for the UBC1-EA strain and wildtype. D. A flow-chart depicting the sample preparation for a SILAC-based proteomic shotgun experiment aimed to explore changes in protein abundance in Ubc1-EA expressing yeast cells. E. Log2 transformed fold changes of proteins between the UBC1-EA strain and wildtype were determined. The forward and reverse SILAC experiments are shown on the x or y axis, respectively. Genes, which were determined to be significantly different in their protein abundance, are marked in red and labeled. Asterisks (*) indicate genes, which are upregulated at a transcriptional and/or translational level upon heat stress A double cross (‡) marks genes, for which the protein product changes in abundance and/or localization in response to DNA replication stress F. Ubc1-EA was genomically integrated into various yeast strains. Exponentially growing cells of the indicated genotype were spotted onto YPD plates in serial 10‑fold dilutions. Where noted cells were incubated at elevated temperature (37 °C) or in presence of hydroxyurea (HU) to induce DNA replication stress.
Figure 2Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. ShRNA targeting Rgcc and control scramble-shRNA were nucleofected separately. Ki67 were co-stained with GFP. To quantify the ratio between NSC markers and GFP positive cells, only the cells in VZ and SVZ are taken into account. And ROIs of 800*1900 pixel were randomly picked for counting. Scale bars: 20 μm. Quantification of the relative ratio of Ki67 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. Sox2 were co-stained with GFP. Scale bars: 20 μm. Quantification of the relative ratio of Sox2 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. Pax6 were co-stained with GFP. Scale bars: 20 μm. Quantification of the relative ratio of Pax6 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 5 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E12.5. All the brains were harvest at E17.5. Tbr1 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along Tbr1 positive region for counting. Scale bars: 100 μm. Quantification of the relative ratio of Tbr1 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E13.5. All the brains were harvest at E17.5. Ctip2 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along Ctip2 positive region for counting. Scale bars: 100 μm. Quantification of the relative ratio of Ctip2 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E13.5. All the brains were harvest at E17.5. Satb2 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along ventricle for counting. Scale bars: 100 μm. Quantification of the relative ratio of Satb2 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. Scale bars: 100 μm. The ratio of GFP positive cells in VZ/SVZ, IZ, and CP were counted for migration analysis.
Figure 2(A) Z-Projection of confocal image stacks spanning 5 μm of a control embryo (left) co-expressing endogenously tagged mVenus::βH-spectrin (green) and the plasma membrane marker GAP43::mCherry (magenta) and of an embryo additionally expressing a maternally driven anti-GFP nanobody KD module to knock-down βH-spectrin (right). Co-expression of the anti-GFP nanobody KD system resulted in a drastic decrease of βH-spectrin protein levels as assessed by the fluorescent signal intensity which dropped below the detection limit. (B) Pie diagrams quantifying the percentage of control embryos (top, n=50) and βH-spectrin knock-down embryos (bottom, n=40) that formed ventral furrow either normally or with minor phenotypes (green) and the percentage of embryos that displayed severe abnormalities or did not at all undergo tissue invagination (red). The assessment of phenotypes was done in an unbiased randomized approach. (C-J) Confocal images of a control embryo (C,D) and βH-spectrin knock-down embryos with a minor phenotype (E,F), with a severe phenotype that underwent tissue invagination (G, H) and with a severe phenotype that did not undergo tissue invagination (I, J). Surface view is shown in (C,E,G,I) and the cross section through the tissue at two different time points in (D,F,H,J). All embryos expressed the plasma membrane marker GAP43::mCherry. Scale bars, 20 μm. (K, L) Quantification of apical surface area (K) and anisotropy (L) in control embryos (green, nControl: 6 embryos) and in βH-spectrin knock-down embryos (red, nβH-spectrin KD: 7 embryos). Solid lines indicate the mean values and the semi-transparent regions the corresponding standard deviation.
Systemic targeting of orthotopic glioblastoma with RGD4C/AAVP-Grp78-HSVtk.Figure 3Immuofluorescent staining for αv integrin expression in brain sections including intracranial tumor (T) and the surrounding healthy brain (B). Scale bar, 100μm. Co-staining of CD31 (red) and phage (green) in brain sections comprising tumor and healthy brain. Scale bar, 100μm. A high-magnification view from the low-magnification insert of the tumor section is shown. Scale bar, 25 μm. Quantification of the relative homing ability of RGD4C/AAVP-Grp78-HSVtk to brain tumors, brain tissue and pancreas after intravenous administration. The data were normalized both to non-targeted vector and to tissue weight then expressed as relative TU. Data shown are representative of two experiments, n=5. One-way ANOVA with Tukey's multiple comparison test (GraphPad Prism 6) was used for data analysis. Quantification of the vector dose reaching orthotopic GBM in mice upon intravenous administration of 5x1010 TU of RGD4C/AAVP vector. The vector amount, in tumors, was expressed as TU/g tumor tissue. Data shown are representative of two experiments, n=5.
Figure 5(A) Glycan microarray analyses of mClec4g, mCD209c and hCLEC4G. Terminal GlcNAc, Gal or Man are colour-coded along the X-axis. Miscellaneous epitopes could not unambiguously be grouped to either terminal GlcNAc, Gal or Man. Mucin type O-glycans, characterized by a reducing-end GalNAc, are highlighted. The identified main binding epitope of each lectin is shown in red. (B) 3D structural modelling of glycosylated trimeric Spike (green with glycans in yellow) interacting with glycosylated human ACE2 (purple with glycans in salmon). The CRD of hCLEC4G (cyan with Ca2+ in orange) was modelled onto Spike monomer 3 glycan site N343 (complex type glycan with terminal GlcNAc in purple-blue) and the CRD of hCD209 (dark blue with Ca2+ in orange) was modelled onto the Spike monomer 3 glycan site N234 (Oligomannose structure Man9 in red). Structural superposition of CLEC4G and ACE2 highlights sterical incompatibility.
Figure 1Population analysis. Pre-enriched iNKT cells from WT BALB/c mice (NKT1, Red; NKT2, Black) were stained with Indo-1. As a measure of intracellular [Ca2+], the ratio of Indo (Violet) and Indo (Blue) was analyzed by flow cytometry. Timing of the addition of cross-linker, goat anti-Armenian hamster IgG (H+L), and extracellular calcium (mM) was as indicated; Single cell analysis. Average profiles of Ca2+ influx induced by anti-CD3ε, and anti-CD28 in freshly sorted individual NKT1 (Red) and NKT2 (Black) cells, labeled by Fura 2. The average signal from more than 200 cells is shown. The timing of antibody addition, and the extracellular [Ca2+] are indicated (mM). Pre-enriched thymocytes from WT BALB/c thymi as in (a) were labeled by CFSE (CFSE+), mixed with pre-enriched thymocytes from SKG thymi (CFSE-), loaded with Indo-1, and stimulated as in (a). Ratio of Indo (Violet) and Indo (Blue) was analyzed by flow cytometry. Comparison of cytoplasmic Ca2+ profiles between WT and SKG NKT1 (c) and NKT2 (d) cells in the same test tube are shown. Data are representative from one of at least three independent experiments. Graphs represent the average Ca2+ levels.
Figure 2Top, left: Schematic representing the conditions utilized for transcriptomic analyses (n=2) of HT-sensitive LNCaP cells. Cells were deprived of hormones for 72 hours, followed by either treatment with 2.5uM veliparib (PARPi) or vehicle control (DMSO) for 1 hour, then subsequently treated with either 1nM DHT or vehicle control (EtOH) for 16 hours. Top, right: Immunoblot with the indicated antisera. Bottom: Volcano plots of transcripts found to be differentially regulated by DHT v. EtOH (left) or DHT v. PARPi followed by DHT (right). Red dots indicate transcripts that were both statistically significantly altered (p<0.05) and more than 1.5-fold changed Top, left: Schematic representing the conditions utilized for transcriptomic analyses (n=2) of CRPC C4-2 cells. Cells were deprived of hormones for 72 hours, followed by either treatment with 2.5uM veliparib (PARPi) or vehicle control (DMSO) for for 16 hours. Top, right: Immunoblot with the indicated antisera. Bottom: Volcano plots of transcripts found to be differentially regulated PARPi v. vehicle control. Red dots indicate transcripts that were both statistically significantly altered (p<0.05) and more than 1.5-fold changed Genes found to be down-regulated by PARPi as described above (p value < 0.05, 1.5-fold change) in either HT-sensitive cells (left) or CRPC cells (right) were queried against the expression of these genes in the Grasso et al data set in Oncomine. Benign = grey, primary PCa = blue, metastases = orange. Boxplot was generated using the mean expression of the PARPi down-regulated genes in the indicated data sets. Statistical significance determined by two-tailed Student"s t
Nuclear shape regulates transcription factor activity.Figure 2Analysis of transcription factor activity (Affymetrix combo protein-DNA array) for Ctrl, Blebb and Blebb + AP conditions. Each square corresponds to a specific transcription factor (TF) activity and is color coded to show the relative activity. ① To identify TFs regulated by nuclear shape, we selected TFs that were more active in Ctrl and Blebb + AP compared to blebbistation-treated cells (Blebb). TF's target genes and GAPDH mRNA levels were analyzed by real-time qPCR. Results are expressed as relative mRNA expression levels. TEAD activity was analyzed in HeLa cells co-transfected with a renilla plasmid as a luciferase reporter plasmid controlled by the TEAD-responsive promoter, and with a renilla plasmid as a gene reporter; HeLa cells were cultured on 1 and 50 kPa (n = 3, t-test ***p<0.01) or treated with blebbistatin and blebbistatin + AP (n = 6; t-test *p<0.5). AP1 activity was assessed in the indicated conditions using AP1 reporter assay. Heatmap of mRNA expression profiles of genes regulated by TEAD and AP1.
Comparison of the GI50 values of RK-33 with FDA-approved drugs on the NCI-60 panel of cell lines.Figure 4A, B The graph depicts the growth inhibitory properties (GI50) of RK-33 for the NCI-60 panel of cell lines. The NCI-60 is a panel of 60 extensively characterized human cell lines derived from nine distinct tumor types: melanoma, ovarian, renal, breast, leukemia, lung, prostate, colon, and CNS. C Network analysis of 102 FDA-approved drugs and RK-33 based on GI50 in the NCI-60 cell line panel. D Unsupervised cluster analysis of the 102 FDA-approved drugs based on the correlation structure of the GI50 levels. The result is shown as a symmetric heat map with positive associations depicted in yellow and negative associations shown in blue.