Journal: Nature

Article Title: Response and resistance to BET bromodomain inhibitors in triple negative breast cancer

doi: 10.1038/nature16508

Figure Lengend Snippet: a, Cellular viability of SUM159 and SUM159R cells transfected with siRNAs targeting bromodomain proteins. *indicate statistical significance (paired t-test) of the marked differences as follows: SUM159: siBRD2 vs. siBRD3, p=0.013, siBRD3 vs. siBRD4, p=0.0154 and SUM159R: siBRD2 vs. siBRD3, p=0.0159, siBRD2 vs. siBRD4, p=0.0048; siBRD3 vs. siBRD4, p=0.0068. b , Cellular viability of SUM159R cells expressing TET-inducible BRD4-targeting or lacZ shRNAs. All error bars represent SEM. P-values indicate statistical significance of the observed differences (unpaired t-test). c, Boxplot showing the log2 fold change in H3K27ac genomic occupancy at regions bound by Bio-JQ1 in parental SUM159 or resistant SUM159R cells. d, Heatmap showing the expression of genes that are up or down regulated by JQ1 versus DMSO after 24 hours treatment in parental SUM159 cells. Each row shows the expression of a single gene in either DMSO or JQ1 treated cells at 24 hours after treatment in SUM159 cells (left four columns) or SUM159R cells (right four columns). Expression values are colored according to fold change relative to the median for each row. Genes are ordered by fold change +/− JQ1 24 hours after treatment. e,f, Boxplots showing the log2 fold change in expression at genes that are up ( e ) or down ( f ) regulated by JQ1 versus DMSO after 24 hours of treatment in parental SUM159 cells. Log2 fold change in expression is shown for either parental SUM159 or resistant SUM159R cells. g , Boxplots showing expression of genes that are up or down regulated by JQ1 versus DMSO after 24 hours of treatment in parental SUM159 cells. Expression is shown in DMSO and JQ1-treated conditions in units of FPKM for either parental SUM159 (left) or resistant SUM159R (right) cells. The statistical significance of the difference between gene expression distributions for SUM159 DMSO and JQ1 treated cells is shown (p <0.01). The difference between all other distributions are considered non significant (N.S). The statistical significance of the difference between SUM159 DMSO gene expression distribution and all other distributions is shown (* p-value < 1e-3). The difference between all other distributions are considered non significant. h , Examples of luminal and basal cell-specific genes, and MYC in SUM159 and SUM159R cells. RNA-seq tracks are shown. i , H&E staining and immunofluorescence analysis of luminal (CK18 and LMW) and basal (CK17 and HMW) cytokeratins and luminal (VIM and CD24) and basal (CDH1, CD44, and pSTAT3) cell markers in SUM159R xenografts. All error bars represent SEM. Scale bars show 100μm for H&E and 50μm for IF respectively.

Article Snippet: Antibodies used for immunoblotting, immunoprecipitation and ChIp-seq were as follows: BRD4 (Bethyl, A301-985A), MED1 (Bethyl, A300-793a), BRD3 (Bethyl, A302-368A), BRD2 (Bethyl, A302-583A), MYC (Santa Cruz, sc764), p-STAT3 (Cell Signaling, 9145S), STAT3 (Cell Signaling, 4904), p-STAT5 (Cell Signaling, 9351), p-JAK2 (Cell Signaling, 3771), CYCLIN D1 (Cell Signaling, 2922), p-H3 (Cell Signaling, 12201), CK2 substrate (Cell signaling, 8738), PP2A-A (Cell signaling, 2039), PP2A-C (Cell signaling, 2038) and p-BRD4 was a gift from Dr. Chiang at University of Texas Southwestern Medical Center.

Techniques: Transfection, Expressing, Gene Expression, RNA Sequencing, Staining, Immunofluorescence