Open Access Highly Accessed Research article

Ligand-free estrogen receptor activity complements IGF1R to induce the proliferation of the MCF-7 breast cancer cells

Anne-Marie Gaben12*, Michèle Sabbah12, Gérard Redeuilh12, Monique Bedin12 and Jan Mester12

Author Affiliations

1 Inserm U938, Centre de Recherche Saint-Antoine, Hôpital Saint-Antoine, Bâtiment Kourilsky, 34 rue Crozatier, 75571, Paris cedex 12, France

2 Université Pierre-et-Marie-Curie Paris 6, 75005, Paris, France

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BMC Cancer 2012, 12:291  doi:10.1186/1471-2407-12-291

Published: 16 July 2012

Additional files

Additional file 1 Figure S1:

Knock-down of the Akt signal by shAkt. Transfections were carried out by the Icafectin method (Eurogentec) according to the manufacturer’ protocol with the shRNA as indicated. The cells were serum-starved during 48h and then harvested and analyzed by Western blotting with the Akt antibody. Actin was used as control. (PPT 114 kb)

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Additional file 2 Figure S2:

p- Akt signal is abolished by phosphatase. The cells were starved as in Figure 2 and then stimulated by addition of insulin (1 mM) for 1 h. The cells were lysed in a buffer without EDTA and proteases inhibitors. Portions of lysates (200 μg of total protein) were incubated with calf intestinal alkaline phosphatase (0.05 U/mg protein) for 1 h at 37°C. The lysates were analyzed by Western blotting for Phospho Ser473-Akt. (PPT 114 kb)

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Additional file 3 Figure S3:

Serum-deprived MCF-7 cells do not secrete autocrine factors. The cells were made quiescent in medium with ICI 182780 during 48 h. They were then placed for 6 h in fresh medium (serum- and phenol red-free) with ICI 182780; this was used as conditioned medium (CM). Another series of dishes were stimulated for 1 h or 3 h with CM or with insulin as a positive control, lysed and analyzed for phospho-Ser473 Akt. (PPT 100 kb)

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Additional file 4 Figure S4:

Cell cycle progression of cells stimulated by insulin or E2. MCF-7 cells were deprived of serum in phenol red-free medium with or without ICI 182780 during 48 h, and then stimulated with insulin or with E2 as described in the text. Cells harvested at the different time points were labeled with propidium iodide and analyzed by flow cytometry. The data were evaluated using the ModFit LT software. (PPT 196 kb)

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Additional file 5 Figure S5:

Akt phosphorylation is equally induced by IGF-I and insulin in cells exposed to ICI 182780. Serum- and E2-starved cells exposed or not to ICI 182780 during 48 h were stimulated with IGF-I (10 nM) or insulin (1 mM) for 1 h. The lysates were analyzed for phospho- Ser473 Akt. (PPT 297 kb)

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