Supplementary Figure 1 -
levels of Aurora-A transcripts in 17β-estradiol-treated cells. Higher Quantitative real-time PCR were performed on RNA isolated from MCF7 cells at different time points after estradiol treatment. The results are presented as the ratio of Aurora-A levels to 18S RNA and are the mean of two independent experiments.
Supplementary Figure 2 -
E2 promotes cell proliferation. (a) Cell proliferation was assessed at 24 h after E2-treatment by BrdU incorporation and compared with the control vehicle-treated cells. Nuclei were stained with propidium iodide. (b) Two hundred nuclei were counted for each sample and the percentage of BrdU-positive cells were presented as bar diagram. The results presented are the mean of two independent experiments.
Supplementary Figure 3 -
Knockdown of Aurora-A impedes growth MCF7 cells in a dose-dependent manner. MCF7 cells were transfected with either 10 nM control siRNA or 1-10 nM Aurora-A specific siRNA. Twenty-four hours post-transfection, 10nM E2 was added and cells were allowed to grow for further 72h. Control cells were maintained in stripped medium in the absence of E2. Medium with and without E2 was replaced every 24h. After 72h hormonal treatment, viable cells were counted by tryphan blue dye exclusion. Mock-treated sample without siRNA and E2. treatment was taken as 100%.
Supplementary Figure 4 -
Dose-dependent effect of Aurora-A knockdown on the extent of apoptosis. MCF7 cells were transfected with either 10nM siGL2 or different concentrations (2.5 and 10nM) of siAurora-A. After 8h transfection, cells were treated with or without 10nM E2 for up to 72h. Floating and adherent cells were collected and stained with Annexin V-FITC (BD Biosciences Pharmingen) and propidium iodide (PI) according to the manufacturer’s instruction. Cells were analyzed immediately after staining by using FACSCalibur flow cytometry and CellQuest software (BD Biosciences). The results represented are the average of two independent experiments.
Supplementary Figure 5 -
Knockdown of Aurora-A decreases the percentage of cells in S phase. MCF7 and IBEP-2 (a kind gift from Dr Guy Laurent, University of Mons-Hainaut, Belgium) cells were transfected with either 10nM siGL2 or different concentration of siAurora-A. Eight hours after transfection, cells were grown in regular RPMI media for 72h. Cells were collected and fixed with 70% ethanol overnight at 4°C. The fixed cells were analyzed for their cell cycle profile by flowcytometry. The numbers provided are the percentage of cells at different phases of the cell cycle and are average values from two independent experiments.
