Transient Elevation of c-Jun and phospho-c-Jun by PKCη and PKCδ in TPA-treated H9 cells.

<p>H9 cells (A) and their subclones stably transfected with shRNAs against either PKCδ (B) or PKCη (C), were treated with TPA in absence (rows 1 to 3) or presence (rows 4 to 6) of the PKC inhibitor BI. Aliquots of the whole-cell extracts, prepared at the indicated times of the TPA ± BI treatment, were subjected to Western blot analysis with monoclonal antibody against c-Jun (rows 1 and 4). Then the blots were re-processed with anti phospho-c-Jun antibody (rows 2 and 5). Equal sample loading was assessed by re-processing the blotted filter with anti actin antibody (rows 3 and 6). (D) <b>Elevation of c-Jun and phospho-c-Jun by ectopically introduced PKCη and PKCδ</b>. H9 cells were transfected with plasmids expressing constitutively active PKCη or PKCδ in the absence or presence of BI. At 24 hr post transfection the cells were examined for the level of c-Jun and phospho-c-Jun by Western blot analysis as detailed for panels A, B and C. Non-transfected cells served as control.</p

Reciprocal co-immunoprecipitation analysis of phospho c-Jun binding to the Sp1-p53 complex and evidence for its involvement in the delay of the PKC-antagonized LTR activation.

<p>Jurkat (A) and H9 (B) cells were treated with TPA for the indicated times in absence (left panels) or presence (right panels) of BI. Aliquots of their nuclear extracts were immunoprecipitated with mouse antibodies (Mouse IP Ab) against p53 (DO-1) (rows 1, 2, 3), Sp1 (rows 4, 5, 6) and phospho-c-Jun (rows 7, 8, 9) as detailed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029934#s4" target="_blank">Materials and Methods</a>”. The co-immunoprecipitated proteins were dissociated and identified by Western blot analysis with the respective rabbit antibodies (Western Rabbit Ab). To determine whether the phospho-c-Jun binding to the Sp1-p53 complex accounted for the delay of the binding of this complex to ERR-1, c-Jun was knockdown by shRNA in Jurkat (panel C; Jurkat/c-Jun shRNA) and H9 (panel D; H9/c-Jun shRNA) cells. These cells were treated with TPA for the indicated times and their nuclear extracts were examined by EMSA for binding to the 3′-biotin-labeled ERR-1 probe as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029934#s4" target="_blank">Materials and Methods</a>). To explore whether the phospho-c-Jun binding to the Sp1-p53 complex accounted for the delay in the onset of the PKC-antagonized LTR activation by TPA, the Jurkat/c-Jun shRNA cells (panel E) were treated with TPA whereas H9/c-Jun shRNA were pretreated with TPA+BI. Cells without the anti c-Jun served as control. At the indicated times of these treatments the cells were transfected with LTR-Luc. The enzymatic activity was measured at 24 hr post-transfection and plotted as describe in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029934#pone-0029934-g004" target="_blank">Figure 4B</a>.</p

The first LTR activation phase in TPA-treated H9 cells is mediated by the non-phosphorylated c-Jun.

<p>H9 cells and their subclones stably transfected with c-Jun shRNA (H9/c-Jun shRNA) or with p53 shRNA (H9/p53 shRNA) were treated with TPA for 36 hr (the peak time-point of the first phase). (A) Whole cell extracts were prepared from part of the treated cells for measuring the level of the non-phosphorylated c-Jun (c-Jun) by Western blot analysis with anti c-Jun antibody. Equal sample loading was assessed by re-processing the blotted filter with anti actin antibody. TPA-untreated H9 cells without shRNA served as negative control, whereas TPA-treated H9 cells without shRNAs served as positive control. (B) The remaining cells were transfected with the LTR-Luc reporter. TPA-untreated cells without shRNAs served as negative control and TPA-treated cells without shRNAs served as positive control. The pRL-renilla plasmid was included in this and all the subsequent transient transfection experiments as internal control for assessing variation in the transfection efficiency. The enzymatic activities were measured at 24 hr post transfection and the Luc activity was normalized to that of renilla and plotted as fold of the respective control. The documented results presented the average of triplicate transfections ± SE. (C) H9 cells without (left panels) and with (right panels) anti c-Jun shRNA were transfected with plasmid expressing non-phosphorylated c-Jun (ectopic c-Jun) or with plasmid expressing constitutively active PKCδ which elevates only phospho-c-Jun. Cells without these ectopic plasmids served as control. At 24 hr after transfection the whole cell extracts of the transfected and non-transfect cells were subjected to Western blot analysis with anti-c-Jun antibodies (top panels) and with antibodies detecting only phosphorylated c-Jun (middle panels). Equal sample loading was assessed with anti actin antibody (bottom panels). (D) H9 cells (left panel) and their subclone stably transfected with anti c-Jun shRNA (H9/c-Jun shRNA, right panel) were transiently transfected with LTR-Luc alone (control) or together with ectopic c-Jun- or ectopic PKCδ- expressing plasmids. Calculation of the enzymatic activities and their presentation were as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029934#pone-0029934-g004" target="_blank">Figure 4B</a>.</p

Binding of c-Jun and CREB of the TPA-treated H9 cells to TRE III.

<p>(A) Schematic illustration of the U3 region of the LTR. In addition, this scheme illustrates the employed probes carrying the wild type (w.t.) and the mutated TRE III sequences. (B) Aliquots of nuclear extracts prepared from H9 cells at the indicated times of the TPA treatment were analyzed by electrophoretic mobility shift assay (EMSA) for binding to 3′ biotin labeled oligonucleotide probe carrying the w.t. TRE III sequence. (C) The specificity of this binding was assessed with nuclear proteins of the cells prepared after treatment with TPA for 36 hr. This was done by competition with 50 fold molar excess of unlabeled oligonucleotides carrying the w.t (left lane) versus the mutant TRE III (right lane) sequences. (D) The nuclear proteins of the 36 hr TPA-treated H9 cells that bound to the TRE III probe were identified by supershift analysis with the indicated doses of antibodies against CREB, ATF-1, ATF-2, c-Jun and phospho-c-Jun. (E) BI effect on the nuclear protein binding to TRE III was determined by testing it the nuclear extracts of the H9 cells treated for 36 hr with TPA±BI. (F) H9 cells (control) and their sub-clones carrying either the anti CREB or anti c-Jun shRNA were treated with TPA for 36 hr. Then their nuclear extracts were subjected to EMSA with the labeled TRE III probe. (G) Illustration of the knockdown efficiency of CREB (upper blot) and phospho/none-phospho c-Jun by their specific shRNA (lower blot) in H9 cells treated with TPA for 36 hr.</p

Schematic summary of the TPA-induced LTR activation pathways in (A) H9 and (B) Jurkat cells.

<p>Schematic summary of the TPA-induced LTR activation pathways in (A) H9 and (B) Jurkat cells.</p

DNA-protein pull-down and Chromatin immunoprecipitation (ChIP) analyses of binding of nuclear proteins of TPA±BI treated H9 cells to TRE III.

<p>(A) For the DNA-protein pull-down analysis, nuclear extracts were prepared from H9 cells after treatment with TPA±BI for the indicated times. Aliquots (200 µg protein) of these extracts were reacted with 6 µg of 3′-biotin-labeled TRE III probe and further processed as detailed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029934#s4" target="_blank">Materials and Methods</a>”, using the indicated antibodies for identifying the specifically bound proteins. (B) The same analysis was performed as in panel A except that limiting dose (1 µg) of the 3′-biotin-labeled probe was employed in order to check for binding competition between c-Jun and CREB on the limited available AP1 site. (C) The same extracts were subjected to Western blot analysis for assessing the input of all the proteins tested in the experiment of panels A and B. To visualize an intracellular recruitment of nuclear proteins of H9 cells to TREs of a putative integrated HTLV-1 viral genome during the first and the second phases of the TPA-induced LTR activations we employed the ChIP procedure described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029934#s4" target="_blank">Materials and Methods</a>”. This was done with chromatins derived from H9 cells carrying stably transfected LTR-Luc plasmid (H9/LTR-Luc), that were exposed to TPA for 36 hr (the peak time-point of the first phase) or for 96 hr (the second phase). (D) These cells were assessed, first, for the activation of the integrated LTR-Luc in both time-points of the TPA treatment (right and middle panels). Untreated cells served as control (left column). The depicted results represent the average of the triplicate assays ± SE. Then the chromatin of the cells treated with TPA for 36 hr (panel E) and 96 hr (panel F) was subjected to ChiP analysis for binding of CREB, c-Jun, ATF-1, ATF-2 and phospho-c-Jun using the indicated doses of the corresponding precipitating antibodies.</p

Binding of the Sp1-p53 complex to the Sp1 site of the ERR-1 in TPA ± BI treated H9 cells.

<p>(A) Schematic illustration of the ERR-1 probe sequene: The upper probe carries the wild type sequence whereas the lower probe is mutated by 3 substituted nucleotides within the Sp1 binding site depicted in small underlined letters. (B) H9 cells were treated with TPA ± BI for the indicated times and aliquots of their nuclear extracts were tested for binding to 3′ biotin-labeled w.t. ERR-1 probe by EMSA. (C) The specificity of this protein binding to the ERR-1 probe was determined in nuclear extract of cells treated with TPA for 96 hr (i.e. at the second phase of the LTR activation). This was done by competition with 50 molar excess of unlabeled oligonucleotide carrying the w.t. or the mutated ERR-1 sequence. For additional control we employed the same excess of the oligonucleotide carrying the TRE III sequence (shown in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029934#s4" target="_blank">Materials and Methods</a>” and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029934#pone-0029934-g002" target="_blank">Figure 2A</a>) as an irrelevant competitor. (D) The proteins bound to the ERR-1 probe, were identified by supershift analysis of the nuclear extract derived from H9 cells treated with TPA for 96 hr using 1, 2 and 4 µg ofthe following antibodies: anti albumin (as an irrelevant negative control), or with anti Sp1 and the DO-1 anti p53 (which detect both the w,t. and mutant p53 proteins). A combination of anti Sp1 and anti p53 antibodies (1 and 2 µg of each of them) was employed in the last 2 lanes at the right side of the blot, whereas no antibody was employed in the control cells presented in the first lane at the left side of the blot. (E) To determine whether the Sp1-p53 complex that bound to the ERR-1 probe contained w.t. or mutant p53 protein, a similar supershift analysis was performed with pAb1620 antibody which identified only w.t. p53 and pAb240 antibody that identified only mutant p53. (F) To demonstrate the intracellular recruitment of these nuclear proteins to the ERR-1 site in LTR integrated within the cellular genome, we carried out chromatin immunoprecipitation (ChIP) analysis in the H9 cells stably transfected with LTR-Luc (H9/LTR-Luc) which were treated with TPA for 96 hr. The immunoprecipitation was performed with 2, 4 and 8 µg of each of the indicated antibodies. (G) Jurkat/LTR-Luc cells were tested for activation of their integrated LTR-Luc by 48 hr-TPA-treatment. Untreated Jurkat/LTR-Luc cells served as control. (H) Jurkat (left panel) and H9 (right panel) cells were co-transfected with the pG13-Luc reporter and BRCA1-expressing plasmid in the absence and presence of anti p53-shRNA. Cells without BRCA1 expressing plasmid served as basal control.</p

Role of PKCα and PKCε in the phospho-c-Jun elevation in TPA-treated Jurkat cells.

<p>(A) Jurkat cells were treated with TPA in absence (rows 1–3) or presence (rows 4–6) of the inhibitor BI. Aliquots of the whole-cell extracts, prepared from the cells at the indicated times of the TPA± BI treatments, were subjected to Western blot analysis, first with anti c-Jun, then with anti phos-c-Jun and finally with anti actin antibody as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029934#pone-0029934-g001" target="_blank">Figure 1</a>. Panel (B) shows the efficiency of the specific shRNA-mediated knockdown of the indicated PKCs isoforms to be employed in the experiments illustrated in the next panels. Jurkat subclones stably transfected with anti PKCα (B) or anti PKCε (C) shRNAs or with both of them (D) were treated with TPA± BI for the indicated times and then examined by Western blot for the level of phos-c-Jun and actin panel (A). As negative control, similar analyses were performed with Jurkat cells transfected with anti PKCη (F) or anti PKCδ (G) shRNAs. To quantify the effect of these knockdowns on the level of the tested c-Jun and phosho-c-Jun we performed densitometry measurements of the bands in the original exposed films of the Western blots. The results are presented as % of the largest band in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029934#pone-0029934-g001" target="_blank">Figure 1A</a> row 1 which was designated as 100%. To assess the knockdown effects of the employed shRNAs on their target PKC isoforms (G), whole cell extracts of Jurkat cells without (left) or with (right) the specific shRNA against the indicated PKCs were subjected to Western blot analysis with the respective antibodies.</p

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

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