16 research outputs found
pCDNA3-ERĪ± transfectants of MDA-MB-231: Assaying stability of ERĪ± 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 50ā70% 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-ERĪ± transfectants of MDA-MB-231: Testing Response to ERĪ± ligand via the Dual-Luciferase reporter assay.
<p>Clones were plated in 24-well tissue culture plates at 50ā70% 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
pCDNA3-ERĪ± transfectants of MDA-MB-231: Western immunoblot analysis of ERĪ± expressing clones.
<p>The MCF-7 cell line was used as an ERĪ±-positive control.</p
Characterization of ectopically expressed RNAs by long range RT-PCR.
<p>MDA-MB-231 parental cell line (231-parental), its pcDNA3-ERĪ± stable transfectant (ERĪ±-2), and its ERĪ±-IRES stable transfectants (ERĪ±-IRES-5 and ERĪ±-IRES-3) were analyzed for expression of ERĪ±āharboring transcript (1.8 kb), Hygromycin B resistance gene-containing transcript (1.0 kb), and ERĪ±-IRES-Hygro<sup>R</sup> fused transcript (3.2 kb), by RT followed by long range PCR amplification. pERĪ±-IRES DNA served as a PCR positive control for the ERĪ± cDNA primers (1.8 kb), the Hygromycin B resistance gene ORF primers (1.0 kb), and the 5ā² sense ERĪ± primer plus 3ā² antisense Hygro<sup>R</sup> fused ORFs primers (3.5 kb). <b>A</b> First four lanes from left contain the ERĪ± cDNA primers; lanes 5ā8 the 5ā² sense ERĪ± primer together with the 3ā² antisense Hygro<sup>R</sup> gene primer. <b>B.</b> The 5ā² sense ERĪ± primer together with the 3ā² antisense Hygro<sup>R</sup> gene primer. <b>C.</b> Lanes 1 and 2 from left, the ERĪ± primers. Lanes 3 and 4 the Hygro<sup>R</sup> gene primers. Primer sequences are detailed in the ā<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031977#s2" target="_blank">Methods</a>ā section.</p
pERĪ±-IRES MDA-MB-231 transfectants: ERĪ± expression via Western immunoblot analysis.
<p><b>A.</b> MDA-MB-231 clones selected for Hygromycin B resistance were lysed and ERĪ± expression was tested by Western immunoblot analysis. ERĪ± positive MCF-7 cell line was used as a positive control. MDA-MB-231 parental cell-line represented the negative control. <b>B.</b> Representation of ERĪ± steady state expression values. The values of ERĪ± expression were normalized to tubulin expression, with MCF7 value being one unit.</p
pERĪ±-IRES MDA-MB-435 (A) & GILM2 transfectants (B): ERĪ± 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>. ERĪ± values were normalized to the values obtained at each time point with MCF-7 cells, taken as the 100%.</p
pERĪ±-IRES MDA-MB-435 transfectants: Western immunoblot analysis of ERĪ± protein.
<p><b>A.</b> MDA-MB-435 cell clones selected for Hygromycin B resistance were lysed and ERĪ± expression was tested by Western immunoblot analysis. ERĪ± 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 ERĪ± steady state expression values. The values of ERĪ± expression were normalized to Ī± tubulin expression in the cells.</p
pCDNA3-ERĪ± transfectants of MDA-MB-231: Dual-Luciferase reporter assay for ERĪ± activity.
<p>G-418 resistant cell clones were seeded in 24-well tissue culture plates at 50ā70% 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
Map of pIRES-ERĪ± 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
pERĪ±-IRES MDA-MB-231 transfectants: Dual-Luciferase reporter assay for ERĪ± 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