ER-α36 mediates E2 and tamoxifen induced c-Myc expression.

<p>A, Western blot analysis of c-Myc expression in Hec1A/V and Hec1A/RNAi cells treated with 10 nM E2 or 2 µM Tam for 12 h. Levels of expression were normalized to the levels of β-actin, and each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells (−) vs. treatments. #, P<0.05 compared to Tam-treated Hec1A/V cells. B and C, Hec1A cells were treated for 12 h with 10 nM E2 or 2 µM Tam or together with 10 µM of MEK inhibitor U0126 or 50 µM PI3K inhibitor LY294002. Levels of c-Myc expression were normalized to the levels of β-actin, and each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells (−) vs. treatments. #, P<0.05 compared to E2- and Tam-treated Hec1A/V cells.</p

ER-α36 mediates E2 and tamoxifen induced activation of Akt.

<p>Hec1A cells were treated with 10 nM E2 (A) or 2 µM Tam (B) for the indicated time points and the lysates were immunoblotted with an antibody against phosphorylated Akt. Levels of phosphorylation were normalized with the total Akt protein, and each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells. C, Western blot analysis of the Akt phosphorylation in Hec1A/V and Hec1A/RNAi ER36 cells treated with 10 nM E2 or 2 µM Tam for 10 min. Levels of phosphorylation were normalized with the total Akt protein, and each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells. #, P<0.05 compared with E2- or Tam-treated Hec1A/V cells. D, Hec1A cells pretreated with 50 µM PI3K inhibitor LY294002 (LY, Lanes 4, 5 and 6) for 2 h and then treated with 10 nM E2 (Lanes 2 and 5) or 2 µM Tam (Lanes 3 and 6) for 10 min. E, Western blot analysis of Akt phosphorylation in MCF-7/ER36 cells treated with 2 µM Tam for the indicated time points. Expression was normalized to total Akt, and each bar represents mean value ± SEM (n = 3).*, P<0.05 compared for untreated cells. F, Lysates were prepared from MCF-7/ER36 cells treated with DMSO (Lanes 1 and 2), 2 µM of Tam (Lanes 2 and 4) or pretreated with 50 µM PI3K inhibitor LY294002 (LY, Lanes 3 and 4) for 2 h, and immunoblotted with antibodies against phosphorylated Akt or total Akt.</p

ER-α36 mediates tamoxifen induced activation of the MAPK/ERK in Hec1A cells.

<p>A and B, Hec1A cells were treated with 10 nM E2 or 2 µM Tam for the indicated time points. Levels of ERK1/2 phosphorylation were measured in protein extracts with Western blot analysis. Total ERK1/2 was used as loading control. Each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells. C and D, ER-α36 expression in Hec1A/V and Hec1A/RNAi cells. Each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to Hec1A/V cells. E, Hec1A/V and Hec1A/RNAi cells treated with 10 nM E2 or 2 µM Tam were analyzed for the levels of ERK1/2 phosphorylation with Western blot. Total ERK1/2 was used as loading control, and each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells (−) vs. treatments. F, Lysates from Hec1A cells treated with DMSO (Lanes 1, 2 and 3), 10 nM E2 (Lanes 2 and 5), 2 µM Tam (Lanes 3 and 6) or pretreated with 10 µM U0126 (Lanes 4, 5 and 6) for 30 min were analyzed with Western blot analysis.</p

ER-α36 mediates E2 or tamoxifen induced activation of the MAPK/ERK in MCF-7 cells.

<p>A, Western blot analysis of ER-α36 expression in MCF-7/V and MCF-7/ER36 cells. Levels of expression were normalized to levels of β-actin, and each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to MCF-7/V cells. B and C, MCF-7/V cells treated with 10 nM E2 alone or with 2 µM Tam together for the indicated time points. Protein extracts were analyzed with Western blot analysis. Total ERK1/2 was used as loading control. Each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to control cells. D, MCF-7/ER36 cells treated with 2 µM Tam for different time points were analyzed for ERK1/2 phosphorylation with Western blot. Levels of expression were normalized to levels of the total ERK1/2, and each bar represents mean value ± SEM (n = 3).*, P<0.05 compared for untreated cells. E, Lysates were prepared from MCF-7/ER36 cells treated with DMSO (Lanes 1, 2 and 3), 10 nM E2 (Lanes 2 and 5), 2 µM of Tam (Lanes 3 and 6) or pretreated with 10 µM U0126 (Lanes 4, 5 and 6) for 30 min and immunoblotted with antibodies against pERK1/2 or total ERK1/2.</p