Effects of 2500, 2900 and 3400 με on proliferation of primary osteoblast-like cells derived from female and male WT (A), <i>Lrp5</i>^−/− (B) and <i>LRP5</i>^G171V (C) mice.

<p>Changes in absolute number of cells between static and strain of both genotypes and genders are shown. Results are mean ± SEM of three independent experiments. Experiments were repeated 3 times. No significant differences at 2500 and 2900 με were observed. ***p<0.001 and *p<0.05 compared with the static control within the gender. (D) The effects of 3400 με on proliferation of primary osteoblast-like cells derived from female and male <i>LRP5</i>^G171V and <i>Lrp5</i>^−/− mice and their WT littermates. Percentage differences between static and strain of both genotypes and genders are shown. Results are the mean ± SEM of three independent experiments. Experiments were repeated 3 times. There were no significant differences between groups.</p

Cell population doubling time of primary osteoblast-like cells derived from female and male <i>Lrp5</i><b>^−/−</b>, <i>LRP5</i><b>^G171V</b> and their WT littermates.

<p>Doubling time in days between 2 and 8 days of culture of primary osteoblast-like cells isolated from female and male <i>Lrp5</i>^−/−, <i>LRP5</i>^G171V and their WT littermates. Cell doubling time were calculated using GraphPad Prism v5.0 software for Windows (GraphPad Software Inc., San Diego, CA) by nonlinear regression (exponential growth equation) analysis.</p

Percentage of apoptosis in osteoblast-like cells 48 hours after treatment with 0.1% (A), 2.5% (B) and 10% (C) serum.

<p>The TUNEL staining was used to determine the percentage of apoptotic cells in primary osteoblast-like cells derived from male and female WT_HBM, <i>Lrp5</i>^−/− and <i>LRP5</i>^G171V mice. Results are mean ± SEM of three independent experiments. Groups with the same letter are not significantly different.</p

Effects of tamoxifen on cell proliferation of a panel of breast carcinoma cell lines.

<p>MCF-7(LCC1), LCC2, LCC9, R27 and BT474 cells were treated with 0 to 10 µM of tamoxifen for 6 days. Cell proliferation was determined by SRB assay. Points, mean of three independent experiments; bars, SD.</p

Overexpressed active FOXO3a has little effect on cell proliferation and drug sensitivity of the drug resistant breast cancer cell line MCF-7-Dox^R.

<p>MCF-7-Dox^R cells were transiently transfected with the constitutively active FOXO3a(A3) or control vector pcDNA3, A) The transfected cells were analysed by Western blot analysis for FOXO3a and β-actin expression. B) The cells were treated with 0 to 10 µM of doxorubicin for 0, 24, 48 and 72 h and SRB assays were performed on these transfected MCF-7-Dox^R cells after 48 h. The results indicated that the induction of FOXO3a(A3) has little effect on the cell proliferation rate of the drug resistant breast carcinoma cells and in response to doxorubicin treatment.</p

Induction of FOXO3a can confer resistance to the MDA-MB-231 cells.

<p>MDA-MB-231-FOXO3a(A3):ER cells were cultured with 200 nM 4-OHT and collected at 0, 4, 8, 24 and 48 h. A) Total cytoplasmic and nuclear extracts were prepared at the times indicated, separated on polyacrylamide gels, and subjected to immunoblotting with FOXO3a, P-FOXO3a(Thr-32), actin and lamin B1 antibodies. B) SRB assays were performed on the untreated and 4-OHT-treated MDA-MB-231 cells after 48 h of treatment with various concentrations of doxorubicin. The results indicated that the induction of FOXO3a (A3) can protect the drug resistant breast carcinoma MDA-MB-231 cells from the effects of doxorubicin.</p

Representative expression patterns of FOXO3a and P-Akt in tissue microarray.

<p>Tumour tissue samples obtained from breast cancer patients that had been formalin-fixed and paraffin-embedded were immunohistochemically stained with FOXO3a and P-Akt (Thr308) antibodies using the streptavidin-biotin-peroxidase technique. A) Representative FOXO3a staining patterns in both tumour and non-tumour cases (magnification ×170). B) Two representative tumour cases showing corresponding FOXO3a and P-Akt staining patterns (magnification ×170). Case 1 shows high cytoplasmic pAkt staining and strong cytoplasmic and nucleus FOXO3a staining. Case 2 shows weak pAkt and FOXO3a staining.</p

Knockdown of FOXO3a expression decreases Akt phosphorylation and PIK3CA mRNA expression.

<p>BT474 cells were transiently transfected with FOXO3a or control shRNA, and 24 h after transfection cells were analysed by A) Western blot using specific antibodies P-FOXO3a (Thr32), FOXO3a, P-Akt (Ser473), Akt and Actin as indicated and by B) qRT-PCR.</p

Effects of doxorubicin on cell proliferation of a panel of breast carcinoma cell lines.

<p>A) MCF-7 and the derived MCF-7 Dox^R cells were treated with 0 to 10 µM of doxorubicin for 0, 24, and 48 h. B) MCF-7, MCF-7 Dox^R, MDA-MB-231, BT474, and LCC9 cells were treated with 1 µM of doxorubicin for 0, 24, and 48 h. C) MCF-7(LCC1), LCC2, LCC9, R27 and BT474 cells were treated with 1 µM of doxorubicin for 0, 24, and 48 h. Cell proliferation was determined by SRB assay. Points, mean of three independent experiments; bars, SD.</p

Gene-specific primer pairs.

<p>Gene-specific primer pairs.</p