Figure 7

Tamoxifen-insensitivity of RAR expression and the functional categories of target genes of the apo-ER apo-RARα1 axis. (A) Hormone-depleted MCF-7 cells were treated with OH-Tam (100 nM) or vehicle control. Seventy-two hours later, the cell lysates were prepared and analysed by western blot using antibodies specific for RARα, RARβ or RARγ; GAPDH was probed as a loading control. The band intensities are normalized to GAPDH and equalized to a value of 1 for the untreated sample. (B) Real time RT-PCR analysis to confirm the effect of knocking down either ER or RARα on the mRNA levels of representative target genes found by Affymetrix DNA microarray in this study to be regulated by apo-ER and apo-RAR in MCF-7 cells: Cells were transfected with control siRNA, ER siRNA or RARα siRNA and four days later the cells were harvested to extract total RNA for the measurement of the relevant mRNAs; the values were normalized those for GAPDH (control). The P-values for the differences noted in the text were <0.001. (C) and (D) mRNA profiling was performed to identify common target genes of apo-ER and apo-RARα1 in MCF-7 cells. Apo-ER and apo-RARα1 were knocked down separately in hormone-depleted MCF-7 cells. Seventy-two hours after transfection with the appropriate siRNA, total mRNA was extracted and mRNA profiling was carried out using Affymetrixs microarray analysis. P-values for the differences noted in the text were ≤0.001.