Journal: Molecular and Cellular Biology

Article Title: Binding of TATA Binding Protein to a Naturally Positioned Nucleosome Is Facilitated by Histone Acetylation

doi: 10.1128/mcb.21.4.1404-1415.2001

Figure Lengend Snippet: FIG. 2. Hyperacetylation of histones H3 and H4 and binding of TBP within the pS2 proximal promoter in response to the transcrip- tional stimuli estradiol and TPA. (A) Chromatin fragments were im- munoprecipitated with anti-acetylated-histone H3 or H4 antibody as indicated, from human mammary MCF-7 cells treated with the pure antiestrogen ICI 182,780 (lanes 1 and 4), 17b-estradiol (lanes 2 and 5), or 17b-estradiol plus TPA (lanes 3 and 6). DNA purified from these immunoprecipitations was analyzed by quantitative PCR, with the product consisting of nucleotides 2440 to 118 of the pS2 promoter. (B) The same immunoprecipitates described for panel A (same lane designations) were analyzed by quantitative PCR with primer set F from the human beta interferon promoter region (2241 to 21) (41). (C) The relative levels of DNA immunoprecipitated with antibodies against acetylated histones H3 and H4 at the pS2 and beta interferon promoters were quantitated using a PhosphorImager (Molecular Dy- namics). The average amounts of recovered DNA, with standard de- viations, were determined from three independent sets of experiments with separate batches of cells. Levels of DNA immunoprecipitated with anti-acetylated-histone H3 antibodies from ICI-treated cells were similar (within twofold) to background levels of precipitated DNA in the absence of antibodies. Thus, the actual enhancement in association of pS2 proximal promoter DNA with highly acetylated histone H3 may be even more pronounced. (D) Genomic chromatin fragments from MCF-7 cells, treated as described for panel A with either ICI 182,780 (lanes 1, 3, 5, and 7) or 17b-estradiol plus TPA (E2 1 TPA) (lanes 2, 4, 6, and 8), were immunoprecipitated either with anti-TBP antibodies (lanes 5 to 8) or with anti-acetylated-histone H3 antibodies (lanes 1 to 4). 2, absence of E2 1 TPA; 1, presence of E2 1 TPA. Duplicate experimental samples are shown to illustrate reproducibility. The rel- ative levels of TBP binding to the proximal pS2 promoter and of immunoprecipitation with the anti-acetylated histone H3 antibody of this region were analyzed by quantitative PCR. The average increase in TBP binding with transcriptional stimuli was 4.1 6 1.1-fold over back- ground levels, based upon four independent experiments. Quantitative PCR analysis was performed on DNA isolated from input samples prior to immunoprecipitation to confirm equalization of initial cellular material.

Article Snippet: In addition, the antiestrogen ICI 182,780 (Tocris) was added at 2 mM for this 48-h period to the unstimulated samples to fully block ER activation of the pS2 promoter.

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Isolation

Journal: Oncogenesis

Article Title: Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis

doi: 10.1038/oncsis.2015.32

Figure Lengend Snippet: Effects of fulvestrant and 17β-estradiol on expression of the mRNA transcripts for the interaction map nodes in MCF-7 cells. ( a , b ) Cells were exposed to 100 n M fulvestrant or vehicle for 48 h followed by mRNA expression profiling by Affymetrix microarray. Robust Multi-array Average (RMA)-normalized relative mRNA expression of genes induced ( a ) or suppressed ( b ) by fulvestrant are shown; mRNA expression in vehicle-exposed cells is defined as 1.00 for each gene (mean±s.e.m., n ≥5). Asterisk indicates statistically significant changes compared with exposure to vehicle (analysis of variance (ANOVA) * P <0.05). ( c–e ) Cells were exposed to varying concentrations of 17β-estradiol for 48 h followed by Affymetrix transcriptomal profiling. RMA-normalized relative mRNA expression of genes induced ( a ), suppressed ( b ) or unchanged ( c ) by 17β-estradiol are shown. Each datum point represents results of at least three independent experiments (mean±s.e.m.), and asterisk indicates statistically significant changes compared with exposure to vehicle (ANOVA * P <0.05; ** P <0.005).

Article Snippet: Affymetrix microarray analysis (Santa Clara, CA, USA) revealed significant increase in mRNA expression for CAMK1D, CSK, ERBB4 and DAPK3 after exposure of MCF-7 cells to 100 n M fulvestrant for 48 h ( ).

Techniques: Expressing, Microarray

Journal: Cells

Article Title: The G Protein-Coupled Estrogen Receptor (GPER): A Critical Therapeutic Target for Cancer

doi: 10.3390/cells12202460

Figure Lengend Snippet: Modalities and attributes of endocrine treatment for regulatory approved drugs.

Article Snippet: Fulvestrant, an injectable steroidal drug marketed by AstraZeneca as FaslodexTM, is a first-generation SERD, and until very recently, it was the only FDA-approved example of such.

Techniques: Inhibition