Journal: Oncogene

Article Title: CBP/p300 acetyltransferases regulate the expression of NKG2D ligands on tumor cells

doi: 10.1038/onc.2016.259

Figure Lengend Snippet: CBP/p300 were crucial for the expression of RAE-1 in vivo . ( a ) Eμ- Myc CBP/P300 littermates show deletion of either CBP or p300. Genomic DNA extracted from tumor cells of terminally ill Eμ- Myc CBP/P300 double mutants (Bnull) was subjected to PCR using specific primers to detect recombined (Δflox) or non-recombined (flox) genes. Representative examples are shown. ( b ) Flow cytometric analysis of tumor cells from lymph nodes to detect MULT1 and RAE-1 (right panel) and of tumor cells from lymph nodes (tumor), spleen or peripheral blood (PB) (left panel) isolated from Eμ- Myc mice (ctrl) or Eμ- Myc with CBP/p300 -deficient B cells (Bnull). ( c ) Real-time PCR to detect RAE-1 transcripts expressed in tumor cells from Eμ- Myc mice (ctrl) or Eμ- Myc with CBP/p300 -deficient B cells (Bnull). RAE-1 mRNA expression was analyzed relative to HPRT. ( d ) Flow cytometric analysis of MCA-205 cells that were preincubated with 8 μ M C646 and treated with 5 n M LBH589 for 16 h followed by detection of MULT1 and RAE-1 surface expression using flow cytometry.

Article Snippet: The following specific antibodies were used: ac-Lysine (9441, CST, Leiden, Netherlands); MICA (AMO1, BamOmaB); MICA/B (6D4, BioLegend); MICB (BMO2, BamOmaB, Gräfelfing, Germany); MULT-1 (237104, R&D); p-γH2AX (S139, 9718, CST); Rae-1 (R&D, Wiesbaden-Nordenstadt, Germany); ULBP1 (AUMO2, BamOmaB); ULBP2 (BUMO1, BamOmaB); ULBP2 (BAF1298, R&D); ULBP3 (CUMO3 BamOmaB) and isotype controls purchased from BioLegend.

Techniques: Expressing, In Vivo, Isolation, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Nature

Article Title: Epigenetic Silencing by SETDB1 Suppresses Tumor Intrinsic Immunogenicity

doi: 10.1038/s41586-021-03520-4

Figure Lengend Snippet: (a) Proportion of chromatin accessible sites (ATAC-seq) gained in Setdb1 KO LLC or B16 cells that are located within (red) or outside (grey) SETDB1 domains. (b) Proportion of ATAC-seq sites gained in Setdb1 KO LLC or B16 cells that coincide with promoters (light grey), distal TEs (red), or other promoter-distal sites (dark grey). Statistics by permutation testing. (c) Proportion of gained ATAC-seq sites at distal TEs in Setdb1 KO B16 cells that also gain H3K27 acetylation and resemble active enhancers. (d) Coordinate gain of chromatin accessibility and H3K27 acetylation at an example TE-site in Setdb1 KO B16 cells. (e) Activation of genes near (<50kb) gained ATAC-seq sites at distal TEs in Setdb1 KO LLC or B16 cells compared to control genes. Statistics by permutation testing. (f-h) Flow cytometry in control and Setdb1 KO cells showing (f) gating strategy, (g) cell-surface expression (y-axis, median fluorescence intensity (MFI)) for ULBP1 and RAET1 ligands in LLC (left), and MHC-I expression in LLC and B16 (right) +/− induction with IFNγ (10ng/mL, 24hr). Data are mean +/− s.e.m. and reflect 2 independent experiments with 4 biological replicates. Statistics by two-sided Student’s t-test. *P < 0.05; **P < 0.01.

Article Snippet: Cells were stained with hybridoma supernatant against MuLV envelope proteins (ATCC, HB-10392) followed by secondary staining with fluorescent antibodies, or with directly conjugated fluorescent antibodies against ULBP1 (FAB2588P, R&D systems), pan-RAET1 (FAB17582P, R&D systems), or MHC Class I (28–8-6, BioLegend) at 1:100 dilution 50 .

Techniques: Activation Assay, Flow Cytometry, Expressing, Fluorescence

Journal: Nature

Article Title: Epigenetic Silencing by SETDB1 Suppresses Tumor Intrinsic Immunogenicity

doi: 10.1038/s41586-021-03520-4

Figure Lengend Snippet: (a) Tracks show H3K9me3 ChIP-seq, genomic compartments, SETDB1 domains, and genes in control and Setdb1 KO B16 for a 9Mb interval of chr17. Expanded view (below) of the C4a/b locus shows ATAC-seq, RNA-seq, LTRs, and segmental duplications paired by arcs. (b) Enrichment analyses for segmental duplications and TEs within SETDB1 domains (left), and gene-ontology categories within SETDB1 domains overlapping segmental duplications (right). Statistics by permutation and hyper-geometric tests. (c) Basal expression, motif enrichment, and logos for TFs enriched within TE-associated ATAC-seq sites gained in Setdb1 KO cells. Enrichment statistics by binomial test. (d) Genome-wide view (top) shows SETDB1 domains and overlapping segmental duplications (>10kb) exhibiting coordinate activation of genes and TEs upon Setdb1 KO. Expanded views (bottom) show the Ifnz and Ulbp1 loci.

Article Snippet: Cells were stained with hybridoma supernatant against MuLV envelope proteins (ATCC, HB-10392) followed by secondary staining with fluorescent antibodies, or with directly conjugated fluorescent antibodies against ULBP1 (FAB2588P, R&D systems), pan-RAET1 (FAB17582P, R&D systems), or MHC Class I (28–8-6, BioLegend) at 1:100 dilution 50 .

Techniques: ChIP-sequencing, RNA Sequencing Assay, Expressing, Genome Wide, Activation Assay