Figure 8

Effect of depleting RARα1 on cell cycle phase distribution of hormone-depleted T47 D and ZR-75-1 cells. (A) Total RNA was extracted from T47 D and ZR-75-1 cells and normal tissue controls (peripheral blood leukocytes, thymus and spleen) and reverse transcribed into cDNA by RT-PCR. The cDNA fragments were amplified by competitive PCR using forward primers specifically against RARα1 and RARα2 and a common reverse primer. The PCR products were identified as previously described (Figure 5). The cDNA for GAPDH was amplified in each sample as an internal control. In B and C, cells were transfected with control siRNA or RARα siRNA and 72 hours later the cells were harvested to extract total RNA or to prepare cell lysates. In B, the mRNA for RARα1 was measured by real time RT-PCR and the values were normalized those for GAPDH (control). In C, the cells lysates were analyzed by western blot using antibody to RARα; the blots were probed for GAPDH as a loading control. (D) Seventy-two hours after transfection with control siRNA or RARα siRNA, T47 D and ZR-75-1 cells were harvested for flow cytometry analysis to determine their cell cycle phase distribution. P-values for the differences noted in the text were ≤0.001.