Effect of Tax on ERα-CBP/p300 complex binding to BRCA1 promoter.

<p>MCF-7 cells which were or not transfected with 1.5 µg of Tax variants [w.t.Tax, TaxM22, TaxM47 or Tax(V89A)] were treated with E2 at 5 hr before their extraction for examining the binding of ERα, CBP and p300 proteins to BRCA1 promoter by CHIP assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089390#s2" target="_blank">Materials and Methods</a> section. Control cells were not transfected with Tax and not treated with E2. The presented results are an average of three repeated experiments ± SE.</p

Effect of Tax on BRCA1 activation by E2 and 53PB1.

<p>(A) MCF-7 cells were co-transfected with 1.5 µg of BARCA1-Luc reporter and the plasmids expressing 53PB1 and/or Tax. Where indicated, E2 (20 nM) was added to the cultures 5 hr before harvesting the cells for analyzing the reporter expression. (B) and (C) MCF-7 cells were co-transfected with 1.5 µg of BARCA1-Luc reporter and the plasmids expressing different Tax varients without (B) or with (C) E2 treatment. As above, E2 was added 5h before harvesting the cells for Luciferase activity measurement in the cell lysates at 24 h post-transfection. (D) MCF-7 cells were co-transfected with 1.5 µg of the plasmids expressing different Tax variants with E2 treatment and at 24h post transfection the BRCA1 mRNA levels were examined as detailed in “Material and Methods” section. The presented results are an average of three repeated experiments ± SE.</p

Tax physically associates with the ERα-CBP/p300 complex through binding to the recruited CBP/p300.

<p>(A) Schematical model 1 describing the formation of separate ERα-p300/CBP and Tax- p300/CBP complexes in E2 treated breast cells with or without Tax expression. (B) MCF-7 cells were transfected with 1.5 µg of the indicated combinations of w.t.Tax, p300 shRNA, CBP shRNA, p300 and CBP expressing plasmids. The cells were treated with E2 at 5 hr before extracting the cells for coimmunoprecipitation (co-IP) analyses. The whole cell extracts were immunoprecipitated with p300, CBP, ERα and Tax mouse specific monoclonal antibodies as indicated in the figure. The various immunoprecipites were analyzed by Western blot analysis with ERα, p300, CBP and Tax rabbit specific monoclonal antibodies. (C) MCF-7 cells were transfected with 1.5 µg of w.t.Tax or each of its variants V89A, M22 and M47 expressing plasmids. The cells were treated with E2 at 5 hr before extracting the cells for coimmunoprecipitation analyses. The whole cell extracts were immunoprecipitated with Tax mouse specific monoclonal antibody. The various immunoprecipites were analyzed by Western blot analysis with ERα, p300, CBP and Tax rabbit specific monoclonal antibodies. (D) Western blot analysis of the protein expression of ERα, p300, CBP and Tax in the lysates of the cells extracts of all the different transfections in part (B) before co-IP. (E) Schematical model 2 describing the formation of the ERα-p300/CBP-Tax tertiary complex complexe in E2 treated breast cells with Tax expression. (F) Schematical model describing the formation of separate ERα-CBP/p300 and Tax-CBP/p300 complexes_in E2 treated breast cells with Tax and excessive level of CBP/p300 expression.</p

Assessing the expression and functionality of the employed ectopic Tax variants in cancerous and non-cancerous human breast epithelial cell lines.

<p>(A) The tested cells were_transfected with equal doses (1.5 µg) of the Tax varients plasmids and their Tax protein levels were determined at 24 hr post-transfection by Western blot analysis of the whole cell extracts with Tax monoclonal antibody. Equal sample loading was assessed by re-processing the blot with anti actin antibody. The effect of Tax varients on HTLV-1 LTR-Luc reporter (B) and on the NF-κB-Luc reporter (1C) was examined by co-transfecting the appropriate cells with 1.5 µg of either of these repoters and 1.5 µg of each of the tested Tax varients. Luciferase activity was measured in the cell lysates at 24 h post-transfection. The presented results are an average of three repeated experiments ± SE.</p