Hoxa1^WT and Hoxa1^I-V relieve MCF7 cells from contact inhibition, while expressing Hoxa1^WM-AA does not.

<p>MCF7 cells were transiently transfected for Hoxa1^WT, Hoxa1^I-V and Hoxa1^WM-AA, together with Pbx1a and Prep1 cofactors, and grown for three weeks. Controls included cells transfected for the potent oncogene hRAS or cells transfected for Pbx1a and Prep1 only. Foci formation was observed for hRAS, Hoxa1^WT and Hoxa1^I-V transfected cells (arrowheads) while not for CTL and Hoxa1^WM-AA cells.</p

The expression of Hoxa1^WM-AA in human mammary carcinoma cells does not result in increased anchorage independent cell growth.

<p>Cells were grown in soft agar and colonies were revealed by crystal violet staining (A) MCF7-Hoxa1^WT and MCF7-Hoxa1^I-V cells produced a lot of colonies in soft agar while CTL and MCF7-Hoxa1^WM-AA only provide a modest colony growth. (B) For each culture, colonies were counted in three random microscopic fields at 16X magnification. Results were pooled for each type of clones and represented as means ± S.D of triplicates. **, p<0.01; ***, p<0.001.</p

The expression of Hoxa1^WM-AA in human mammary carcinoma cells does not result in increased cell proliferation and growth.

<p>(A) WST-1 based proliferation assays were performed for MCF7-Hoxa1^WT, MCF7-Hoxa1^I-V, MCF7-Hoxa1^WM-AA and MCF7-CTL clones. The proliferation index was determined for each clone as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025247#s4" target="_blank">Materials and Methods</a>. Results were pooled for each type of clones and represented as means ± S.D. of triplicates. *, p<0.05 (ANOVA 2). (B) Cells for MCF7-Hoxa1^WT, MCF7-Hoxa1^I-V, MCF7-Hoxa1^WM-AA and MCF7-CTL clones were inoculated, kept in culture for 16 days and counted after day 4, 7, 9, 11, 14 and 16. Growth curves represent the mean of four independent experiments.</p

Activation of a Hoxa1 target reporter in mammary carcinoma cell clones.

<p>The Hoxa1 target reporter <i>EphA2-r42B-luc</i> is activated in cell clones for Hoxa1^WT and Hoxa1^I-V while not in Hoxa1^WM-AA clones. In each experiment, the pML-EphA2-r42B-luc reporter plasmid was transfected in combination with expression vectors for both Prep1 and Pbx1a. The constitutively active pCMV-LacZ reporter plasmid was added as a transfection control. Results were calculated by a luciferase/β-galactosidase ratio, pooled for each type of clones and represented as means ± S.D. of triplicates. *, p<0.05 and ***, p<0.001 (ANOVA 2).</p

Characterization of MCF7 clones for the constitutive expression of Hoxa1 variants.

<p>(A) Expression of Hoxa1, Neomycin resistance (Neo), Pbx1 and β-actin genes was detected by RT-PCR. While MCF7 cells do not express Hoxa1, clones obtained the stable transfection of Hoxa1^WT, Hoxa1^I-V and Hoxa1^WM-AA coding plasmids express the Hoxa1 variants at similar levels (β-actin used as reference). All cells express the endogenous Pbx1 gene. (B) The Hoxa1 and (C) PBX1B protein immunolocalisation reveals that both proteins localize into the cell nucleus.</p

Transcriptional activity and relative expression of Hoxa1 variants.

<p>(A) The Hoxa1 target reporter <i>EphA2-r42B-luc</i> is activated in MCF7 cells in the presence of expression vectors for Hoxa1^WT, Hoxa1^I-V while not in the presence of Hoxa1^WM-AA or of an empty (CTL) plasmid. In each experiment, the pML-EphA2-r42B-luc reporter plasmid was transfected in combination with expression vectors for both Prep1 and Pbx1a. Results were calculated by a luciferase/β-galactosidase ratio and represented as means ± S.D. of triplicates. ***, p<0.001 (ANOVA2). (B) Detection of Hoxa1 variant proteins from whole cell lysates obtained from transiently transfected MCF7 cells reveal that Hoxa1^WT, Hoxa1^I-V and Hoxa1^WM-AA proteins are equally expressed and stable. No Hoxa1 protein could be detected from MCF7 cells or from cells transfected with an empty (CTL) expression vector. Detection of constitutively expressed β-actin protein was performed as control load.</p