Journal: Oncotarget

Article Title: Generation and characterization of ErbB2-CAR-engineered cytokine-induced killer cells for the treatment of high-risk soft tissue sarcoma in children

doi: 10.18632/oncotarget.19821

Figure Lengend Snippet: Cytotoxic analysis of WT and genetically modified (ErbB2-CAR and mock-vector) CIK cells against ErbB2-expressing (A) RH30 (aRMS) (B) TE671 (embryonal RMS), (C) A204 (embryonal RMS) and (D) RH41 (aRMS) cells using a 3-hour europium release assay are shown. ErbB2-CAR transduction significantly increased the anti-tumor potential of CIK cells against the RH30, TE671, and A204 RMS cell lines within 3-hour co-culture of effector and target cells. Hereby, ErbB2-CAR CIK cytotoxicity correlated with ErbB2 antigen expression levels.

Article Snippet: The aRMS cell lines RH30 and RH41, as well as the embryonal RMS cell lines TE671 and A204, were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany).

Techniques: Genetically Modified, Plasmid Preparation, Expressing, Release Assay, Transduction, Co-Culture Assay

Journal: Oncotarget

Article Title: Generation and characterization of ErbB2-CAR-engineered cytokine-induced killer cells for the treatment of high-risk soft tissue sarcoma in children

doi: 10.18632/oncotarget.19821

Figure Lengend Snippet: Analysis of the cytotoxic capacity of WT and genetically modified (ErbB2-CAR) CIK effector cells against ErbB2-expressing (A) RH30 (aRMS) (B) TE671 (embryonal RMS), (C) A204 (embryonal RMS), (D) RH41 (aRMS), and (E) primary aRMS cells using brightfield imaging cytometry after 16-hour co-incubation of effector and target cells is illustrated. ErbB2-CAR transduction increased the anti-tumor potential of CIK cells against RMS cell lines, as well as against refractory aRMS cell line RH41 (Figure ) and primary RMS cells at very low effector-to-target cell ratios of 10:1, 5:1 and 1:1.

Article Snippet: The aRMS cell lines RH30 and RH41, as well as the embryonal RMS cell lines TE671 and A204, were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany).

Techniques: Genetically Modified, Expressing, Imaging, Cytometry, Incubation, Transduction

Journal: Nature genetics

Article Title: SMARCB1 is required for widespread BAF complex-mediated activation of enhancers and bivalent promoters

doi: 10.1038/ng.3958

Figure Lengend Snippet: BAF47 confers BAF complex stability on chromatin without affecting intra-complex subunit stability. (a) Schematic for rescue experiments in BAF47-deficient cell lines. (b) Nuclear extract inputs and anti-BRG1 IP from G401 nuclear extracts in empty vector and BAF47 conditions. (c) Nuclear extract input and IPs for IgG, BRG1, and BAF47 in control and BAF47Δ/Δ (knockout) HEK293T cells. (d) Silver stain analysis of control IgG and anti-BRG1 IPs in G401 empty vector and BAF47 conditions. (e) anti-BRG1 IP-mass spectrometry proteomics in G401 empty vector and BAF47 conditions for BAF complex subunits. (f-g) Density sedimentation analyses using 10-30% glycerol gradients (10m; 0.5ml/ fx) on nuclear extracts from G401 MRT cells in (f) the empty vector control and (g) BAF47 conditions. (h) (left) Schematic for differential salt extraction experiments in G401 empty vector or BAF47 conditions; (right) Immunoblot analysis of BAF complex subunits in differential salt extraction experiments. (i) Relative densitometry from differential salt extraction demonstrates gained stability of BAF complexes on chromatin in the BAF47 condition. Error bars = mean ± SEM for n=2 biological replicates.

Article Snippet: Of these, four cell lines were purchased from ATCC (G401, G402, A204 and VA-ES-BJ), and three were from RIKEN (HS-ES-1, HS-ES-2R and HS-ES-2M).

Techniques: Plasmid Preparation, Control, Knock-Out, Silver Staining, Mass Spectrometry, Sedimentation, Extraction, Western Blot

Journal: Nature genetics

Article Title: SMARCB1 is required for widespread BAF complex-mediated activation of enhancers and bivalent promoters

doi: 10.1038/ng.3958

Figure Lengend Snippet: Rescue of BAF47 drives a genome-wide gain in BAF complex chromatin occupancy (a) Input blot for TTC1240 and G401 cell nuclear extracts in empty vector and BAF47 conditions. (b) Venn diagram of (left) BRG1 and (right) BAF155 peaks in empty vector and BAF47 conditions in TTC1240 cells. (c) Heatmaps of BRG1 and BAF155 occupancy in TTC1240 empty vector and BAF47 conditions over all BRG1-BAF155 shared sites in the TTC1240+BAF47 condition. (d) Example BRG1, BAF155, and RNA-seq tracks at CDKN1A enhancers in TTC1240 cells. (e) Distance to closest transcription start site (TSS) for conserved (empty-BAF47) and gained (BAF47-only) BRG1-BAF155 sites in TTC1240 cells. (f) Centrimo plots for top four centrally enriched motifs at gained BRG1-BAF155 sites in TTC1240 cells. (g) Average sequence conservation (PhyloP) of conserved and gained BRG1-BAF155 sites. (h) Proliferation analyses of MRT, EpS, and AT/RT cell lines; values shown are relative proliferation between BAF47 and empty vector conditions at noted days. (i-k) Venn diagrams of BRG1 peaks in empty vector and BAF47 conditions in (i) G401, (j) HS-ES-2M, and (k) VA-ES-BJ cell lines.

Article Snippet: Of these, four cell lines were purchased from ATCC (G401, G402, A204 and VA-ES-BJ), and three were from RIKEN (HS-ES-1, HS-ES-2R and HS-ES-2M).

Techniques: Genome Wide, Plasmid Preparation, RNA Sequencing, Sequencing

Journal: Nature genetics

Article Title: SMARCB1 is required for widespread BAF complex-mediated activation of enhancers and bivalent promoters

doi: 10.1038/ng.3958

Figure Lengend Snippet: Resolution of bivalent promoters to activation by BAF complex-mediated opposition of polycomb-mediated repression. (a) Heatmaps of H3K4me3, BRG1, SS18, BAF200, H3K27ac, H3K27me3, and SUZ12 across all hg19 promoters in empty vector condition in TTC1240 cells, ranked by H3K4me3 occupancy. (b) GO term analysis of bivalent genes in TTC1240 cells. (c) Overlap of bivalent genes in G401 and TTC1240 cells. (d) Overlap of BRG1 target genes in empty and BAF47 conditions in TTC1240 cells. (e) Distribution of (left) conserved and (right) gained BRG1 target genes in TTC1240 cells. (f) Overlap of bivalent genes in empty and BAF47 conditions in TTC1240 cells. (g-h) Metagene plots of (g) H3K4me3 and H3K27me3, as well as (h) BRG1, SS18, and BAF200, over all 3512 bivalent promoters in TTC1240 cells. (i) Example tracks at the LAMB1 bivalent promoters demonstrate resolution of bivalent promoters to activation upon gain of BAF complex occupancy in TTC1240 cells.

Article Snippet: Of these, four cell lines were purchased from ATCC (G401, G402, A204 and VA-ES-BJ), and three were from RIKEN (HS-ES-1, HS-ES-2R and HS-ES-2M).

Techniques: Activation Assay, Plasmid Preparation

Journal: Nature genetics

Article Title: SMARCB1 is required for widespread BAF complex-mediated activation of enhancers and bivalent promoters

doi: 10.1038/ng.3958

Figure Lengend Snippet: Collaborative gene activation by BAF complex-mediated enhancer activation and polycomb opposition at bivalent promoters (a) Distribution of significantly-regulated genes in TTC1240 cells. (b) Directional regulation of significantly changed genes in TTC1240 cells, with y-axis indicating proportion of all genes in each category. (c) (left) Overlap of significantly-changed genes in G401 and TTC1240 cell types in empty vector and BAF47 conditions; (right) genes significantly-regulated in both cell lines show significant concordance (p < 2.2e-16, Fisher exact test). (d) Heatmap of 642 significantly changed genes in both G401 and TTC1240 cells. Right bar indicates promoter status of each gene in each cell line using colors from (a). (e) Heatmap of selected genes that are significantly-upregulated by BAF47 in both G401 and TTC1240 cells. (f) GO term analysis of significantly upregulated genes in both G401 and TTC1240. (g) Genes categorized by number of distal gained (BAF47-only) BAF complex sites, broken down by promoter status of genes in each category. n = number of genes in each group. (h-i) Example ChIP-seq tracks of BRG1, SS18, BAF200, H3K27ac, H3K4me3, H3K27me3, and RNA-seq for (h) CTGF and (i) FN1 shows collaborative gene activation via enhancer activation and polycomb opposition at bivalent promoters.

Article Snippet: Of these, four cell lines were purchased from ATCC (G401, G402, A204 and VA-ES-BJ), and three were from RIKEN (HS-ES-1, HS-ES-2R and HS-ES-2M).

Techniques: Activation Assay, Plasmid Preparation, ChIP-sequencing, RNA Sequencing