E2 and IGF-I quickly stimulate GFP-ERα translocation from nucleus to cytoplasm in NWTB3 cells.

<p>ER was mostly located in the nucleus in the non-treated control group, however, only a small part in the cytoplasm after E2 treatment (20 min). ER was significantly increased in the cytoplasm in IGF-I and IGF-I plus ER treatment groups (20 min).</p

Binding of IGF-IR to ERα is stimulated by IGF-I or E2 in NWTB3 cells.

<p>NWTB3 cells transiently transfected with ERα were stimulated with 10 nM IGF-I or 10 nM E2 for 0–4 h. (<b>A</b>) Protein lysates were subjected to either immunoblotting with ERα antibody for determination of the expression of ERα or immunoprecipitated with IGF-IR antibody with subsequent immunoblotting of the precipitated fraction with ERα antibody. (<b>B</b>) The ratio of ERα/IGF-IR rapidly increased from 0 to 4 h, reached peak at 1 h after IGF-I stimulation, meanwhile, E2 treatment also enhanced the ratio with the peak at 1 h. C, NIH3T3 cells without ERα cDNA transfection. <i>N</i> = 3, *, <i>P</i><0.05 as compared to the control groups.</p

ERα potentiates the basal and maximum phosphorylation of ERK1/2 and Akt by IGF-I stimulation.

<p>NWTB3 cells transfected with ERα or vector control were stimulated with 10 nM IGF-I for 0–60 min. (<b>A</b>) Protein lysates were immunoblotted with phospho-ERK1/2, ERK1/2, phospho-Akt, Akt, or ERα antibodies. Transfection of ERα gradually increased phosphorylation of ERK1/2 and Akt after IGF-I stimulation. (<b>B</b>) The ratios of p-ERK1/2 <i>vs</i>. ERK1/2. The maximum phospho-ERK1/2 appeared in 5 min in ERα transfected NWTB3 cells after IGF-I stimulation, while it was 15 min in ERα-negative control cells. (<b>C</b>) The ratios of p-Akt <i>vs</i>. Akt. The maximum of phosphorylation of Akt was at 15 min in ERα-positive cells, but at 60 min in ERα-negative cells. <i>N</i> = 3, *, <i>P</i><0.05 as compared to controls.</p

Cell proliferation is enhanced in ERα-transfected NWTB3 cells by IGF-I stimulation.

<p>To investigate the potential cross-talk between ER and IGF-IR on cellular proliferation, we used NWTB3 cells, which overexpress IGF-IR but are devoid of ER. These cells were transfected with ERα cDNA or vector control for 24 h and subsequently stimulated with 10 nM IGF-I for 48–72 h. In the presence of ER, cell proliferation was significantly increased after 48 and 72 h, respectively, as compared to cells without ER cDNA transfection. <i>N</i> = 3, *, <i>P</i><0.05 as compare to cells without IGF-I treatment; #, <i>P</i><0.05 as compare to the ERα-negative control groups.</p

Binding IGF-IR to ERα is abrogated by IGF-I in MCF-7^SX13 cells.

<p>MCF-7^SX13 cells were stimulated with 10 nM IGF-I for 0–60 min. Protein lysates were subjected to immunoprecipitation with IGF-IR (A) or ERα (B) antibodies with subsequent immunoblotting of the precipitated fraction with ERα antibody (<b>A</b>) or IGF-IR antibody (<b>B</b>), respectively. The ratios of ERα/IGF-IR and IGF-IR/ERα at different time points were similar to each other, <i>N</i> = 3, <i>P</i>>0.05.</p

Binding of IGF-IR to ERα is stimulated by IGF-I in MCF-7 cells.

<p>MCF-7 cells were stimulated with 10 nM IGF-I for 0, 5, 15, 30, or 60 min. Protein lysates were subjected to immunoprecipitation with IGF-IR antibody and subsequently immunoblotted with ERα antibody (<b>A</b>) or immunoprecipitation with ERα antibody and subsequently immunoblotted with IGF-IR antibody (<b>B</b>). The ratio of ERα and IGF-IR rapidly increased from 0 to 15 min, and reached peak at 15 min and gradually decreased from 30 to 60 min (A). Also, the ratio of IGF-IR and ERα rapidly increased from 0 to 15 min, and reached peak at 15 min and gradually decreased from 30 to 60 min (B). <i>N</i> = 3, *, <i>P</i><0.05 as compared to 0 min.</p