Characterization of ectopically expressed RNAs by long range RT-PCR.

<p>MDA-MB-231 parental cell line (231-parental), its pcDNA3-ERα stable transfectant (ERα-2), and its ERα-IRES stable transfectants (ERα-IRES-5 and ERα-IRES-3) were analyzed for expression of ERα–harboring transcript (1.8 kb), Hygromycin B resistance gene-containing transcript (1.0 kb), and ERα-IRES-Hygro^R fused transcript (3.2 kb), by RT followed by long range PCR amplification. pERα-IRES DNA served as a PCR positive control for the ERα cDNA primers (1.8 kb), the Hygromycin B resistance gene ORF primers (1.0 kb), and the 5′ sense ERα primer plus 3′ antisense Hygro^R fused ORFs primers (3.5 kb). <b>A</b> First four lanes from left contain the ERα cDNA primers; lanes 5–8 the 5′ sense ERα primer together with the 3′ antisense Hygro^R gene primer. <b>B.</b> The 5′ sense ERα primer together with the 3′ antisense Hygro^R gene primer. <b>C.</b> Lanes 1 and 2 from left, the ERα primers. Lanes 3 and 4 the Hygro^R gene primers. Primer sequences are detailed in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031977#s2" target="_blank">Methods</a>” section.</p

pCDNA3-ERα transfectants of MDA-MB-231: Assaying stability of ERα trans-activity via the dual luciferase assay.

<p>For each time point, measured in days from the received time of individual clones covering a 60 mm plate, cells were plated in 24-well tissue culture plates at 50–70% density, and grown in DMEM supplemented with 5% FCS. Twenty four hours later, cells were transiently co-transfected with a p2xERE-pS2-luc plasmid together with a pRNL-TK plasmid. Forty eight hours after transfection cells were lysed whereby <i>firefly</i> and <i>Renilla</i> luciferase activities were measured and normalized to the positive control, MCF-7.</p

pCDNA3-ERα transfectants of MDA-MB-231: Testing Response to ERα ligand via the Dual-Luciferase reporter assay.

<p>Clones were plated in 24-well tissue culture plates at 50–70% density under the three growth conditions mentioned in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031977#pone-0031977-g004" target="_blank">Fig. 4</a>. Twenty four hours later cells were transiently co-transfected with a p2xERE-pS2-luc plasmid together with a pRNL-TK plasmid. Forty eight hours after transfection cells were lysed whereby <i>firefly</i> and <i>Renilla</i> luciferase activities were measured and compared to the positive control, MCF-7. MDA-MB-231 parental cell-line was used as a negative control. The presented values were normalized to that of MCF-7 cells seeded in DMEM supplemented with 5% FCS.</p

Map of pIRES-ERα bicistronic plasmid.

<p>The vector contains a single mammalian transcription unit initiating from the CMV immediate early promoter and terminating with an SV40 derived polyA addition fragment.</p

pCDNA3-ERα transfectants of MDA-MB-231: Western immunoblot analysis of ERα expressing clones.

<p>The MCF-7 cell line was used as an ERα-positive control.</p

pERα-IRES MDA-MB-231 transfectants: Dual-Luciferase reporter assay for ERα activity.

<p>Technical details as in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031977#pone-0031977-g006" target="_blank">Fig. 6</a>.</p

pCDNA3-ERα transfectants of MDA-MB-231: Responsiveness to ligand.

<p>MDA-MB-231 derived clones were seeded in 60 mm dishes and grown for 24 hrs under three conditions: DMEM supplemented with 5% FCS, phenol red-free DMEM supplemented with 5% CSS, and phenol red-free DMEM supplemented with 5% CSS and 2×10⁻⁸ M E₂. The top panel shows the 66 KDa ERα protein detected with the anti-hERα antibody. The bottom panel shows the 57 KDa α-tubulin protein within the same blot after stripping the anti-hERα antibody and re-probing with the anti-α-tubulin antibody.</p

pERα-IRES MDA-MB-435 (A) & GILM2 transfectants (B): ERα activity over time.

<p>Technical details as in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031977#pone-0031977-g006" target="_blank">Fig. 6</a>. ERα values were normalized to the values obtained at each time point with MCF-7 cells, taken as the 100%.</p

pERα-IRES MDA-MB-435 transfectants: Western immunoblot analysis of ERα protein.

<p><b>A.</b> MDA-MB-435 cell clones selected for Hygromycin B resistance were lysed and ERα expression was tested by Western immunoblot analysis. ERα positive MCF-7 cell line was used as a positive control. MDA-MB-435 parental cell-line represented the negative control. <b>B.</b> Representation of ERα steady state expression values. The values of ERα expression were normalized to α tubulin expression in the cells.</p

pCDNA3-ERα transfectants of MDA-MB-231: Dual-Luciferase reporter assay for ERα activity.

<p>G-418 resistant cell clones were seeded in 24-well tissue culture plates at 50–70% density in DMEM supplemented with 5% FCS. Twenty four hours later, cells were transiently transfected with the pERE-PS2-luc plasmid together with the pRNL-TK plasmid. Forty eight hours after transfection, cells were lysed whereby <i>firefly</i> and <i>Renilla</i> luciferase activities were measured and normalized to the positive control, MCF7.</p