bcl6 n3 sc 858  (Santa Cruz Biotechnology)

Journal: Oncogene

Article Title: The transcriptional modulator BCL6 as a molecular target for breast cancer therapy

doi: 10.1038/onc.2014.61

Figure Lengend Snippet: BCL6 is expressed in breast cancer. Copy number for BCL6 was analyzed using data from (A) the Cancer Genome Atlas for breast tumors (inset contains percentages) or (B) from the Cancer Cell Line Encyclopedia for breast cancer cell lines. Copy number (C) and BCL6 gene expression (D) from the Cancer Cell Line Encyclopedia for a subset of breast cancer cell lines. (E) Nuclear lysates were generated from the indicated breast cancer cell lines and analyzed for BCL6 expression. Whole cell extracts of Ramos cells served as a positive control for BCL6 expression. PARP served as a control for nuclear protein. (F) Nuclear lysates were generated from the indicated cell lines after 72 hours of transfection with siRNA to BCL6 (or control). PARP served as a control for nuclear protein.

Article Snippet: Cells were fixed in 1% formaldehyde for 10 minutes, sonicated, and lysates were immunoprecipitated overnight with 2 μg of antibodies to BCL6 (sc-858) and RNA polymerase II (sc-9001) from Santa Cruz Biotechnology or acetyl H4 from Cell Signaling Technology.

Techniques: Gene Expression, Generated, Expressing, Positive Control, Control, Transfection

Journal: Oncogene

Article Title: The transcriptional modulator BCL6 as a molecular target for breast cancer therapy

doi: 10.1038/onc.2014.61

Figure Lengend Snippet: BCL6 binds to specific genomic sites in breast cancer cells. (A) ChIP was performed in SK-BR-3 cells for BCL6 binding to target sites identified in lymphoma cells. Data are expressed as BCL6 binding relative to background (at a nonbinding region). (B) De novo motif analysis of genomic sequences associated with BCL6 binding sites in breast cancer cells identified the BCL6 binding motif. (C) Genomic distribution of BCL6 binding sites relative to genes in breast cancer cells. (D) BCL6 ChIP was performed in three different breast cancer cell lines, and binding of BCL6 to binding sites was analyzed by qPCR. (E) Genes containing at least one BCL6 binding site were compared from the breast cancer and lymphoma ChIP-seq datasets.

Article Snippet: Cells were fixed in 1% formaldehyde for 10 minutes, sonicated, and lysates were immunoprecipitated overnight with 2 μg of antibodies to BCL6 (sc-858) and RNA polymerase II (sc-9001) from Santa Cruz Biotechnology or acetyl H4 from Cell Signaling Technology.

Techniques: Binding Assay, Genomic Sequencing, ChIP-sequencing

Journal: Oncogene

Article Title: The transcriptional modulator BCL6 as a molecular target for breast cancer therapy

doi: 10.1038/onc.2014.61

Figure Lengend Snippet: BCL6 modulates gene expression in breast cancer cells. (A) SK-BR-3 cells were transfected with siRNA to BCL6 (or control). Extracts were then analyzed for BCL6 expression by immunoblot (left) or by ChIP for binding of BCL6, RNA polymerase II, and acetylated histone H4 to region B of the BCL6 gene (right). (B) SK-BR-3 cells treated with siRNA to BCL6 (or control) were analyzed by ChIP for BCL6 (top) and RNA polymerase II (bottom) binding at the indicated sites. Binding is expressed relative to a negative control region. (C) Breast cancer cells treated with the indicated expression vectors and siRNA constructs were transfected with a BCL6 responsive luciferase plasmid and analyzed for luciferase activity. (D) SK-BR-3 breast cancer cells treated with siRNA to BCL6 or control were analyzed by qRT-PCR for expression of the indicated genes relative to GAPDH.

Article Snippet: Cells were fixed in 1% formaldehyde for 10 minutes, sonicated, and lysates were immunoprecipitated overnight with 2 μg of antibodies to BCL6 (sc-858) and RNA polymerase II (sc-9001) from Santa Cruz Biotechnology or acetyl H4 from Cell Signaling Technology.

Techniques: Gene Expression, Transfection, Control, Expressing, Western Blot, Binding Assay, Negative Control, Construct, Luciferase, Plasmid Preparation, Activity Assay, Quantitative RT-PCR

Journal: Oncogene

Article Title: The transcriptional modulator BCL6 as a molecular target for breast cancer therapy

doi: 10.1038/onc.2014.61

Figure Lengend Snippet: BCL6 depletion inhibits breast cancer growth and survival. (A) Breast cancer cells were transfected with siRNA to BCL6 and viable cell number was analyzed by measuring ATP dependent luminescence. (B) The indicated breast cancer cell lines were transfected with siRNA to BCL6 or control. BCL6 knockdown was confirmed by immunoblot (top). Viable cell number was measured at 96 hours after transfection (bottom). (C) MDA-MB-468 cells were transfected with the indicated siRNAs, and BCL6 expression (top) and viable cell number (bottom) were measured 48 hours after transfection. (D) MDA-MB-468 cells treated with control or BCL6-targeting siRNA were transfected with expression vectors for BCL6 or STAT3. Expression of the indicated proteins was measured by immunoblot (left), and viable cell number was measured 48 hours after transfection (right). Although the coding sequence of the BCL6 expression construct was wildtype, high efficiency transfection allowed expression in the presence of the siRNA to BCL6.

Article Snippet: Cells were fixed in 1% formaldehyde for 10 minutes, sonicated, and lysates were immunoprecipitated overnight with 2 μg of antibodies to BCL6 (sc-858) and RNA polymerase II (sc-9001) from Santa Cruz Biotechnology or acetyl H4 from Cell Signaling Technology.

Techniques: Transfection, Control, Knockdown, Western Blot, Expressing, Sequencing, Construct

Journal: Oncogene

Article Title: The transcriptional modulator BCL6 as a molecular target for breast cancer therapy

doi: 10.1038/onc.2014.61

Figure Lengend Snippet: BCL6 inhibition decreases the viability of breast cancer cells. (A) MDA-MB-468 breast cancer cells were treated with RI-BPI or control peptide for twelve hours and then expression of BCL6 target genes were analyzed by qRT-PCR. (B) Breast cancer cells were treated with the indicated doses of RI-BPI or control peptide (µM) for 48 hours after which viable cell number was determined. (C) MDA-MB-468 breast cancer cells were treated with 15 µM RI-BPI for 48 hours and then initiation of apoptosis was determined by immunoblot for PARP cleavage. HSP90 served as a loading control. (D) MDA-MB-468 cells were treated with vehicle or 15 µM RI-BPI for 48 hours after which apoptosis was determined by Annexin V/PI staining and flow cytometry. (E) MDA-MB-468 cells treated with control or BCL6-targeted siRNA were then treated with vehicle or RI-BPI for 48 hours and viable cell number was measured. (F) MDA-MB-468 cells were transfected with siRNA to BCL6 and/or Jak2. Relative cell viability was measured after 72 hours. (G) MDA-MB-468 cells treated with control or Jak2-targeted siRNA were treated with 10 µM RI-BPI for 48 hours and viable cell number was determined. (H) MDA-MB-468 cells transfected with siRNA to BCL6 were treated with the Jak2 inhibitor TG101348 (3 µM) for 48 hours and cell viability was measured. (I) MDA-MB-468 cells were treated with 10 µM RI-BPI (BPI) and the indicated doses of TG101348 for 48 hours and then cell viability was measured.

Article Snippet: Cells were fixed in 1% formaldehyde for 10 minutes, sonicated, and lysates were immunoprecipitated overnight with 2 μg of antibodies to BCL6 (sc-858) and RNA polymerase II (sc-9001) from Santa Cruz Biotechnology or acetyl H4 from Cell Signaling Technology.

Techniques: Inhibition, Control, Expressing, Quantitative RT-PCR, Western Blot, Staining, Flow Cytometry, Transfection