Figure 2
9-cis-RA induces the expression of cIAP2 in breast cancer cells in a cell context dependent manner. (A, B, C) Multiplex RNase protections assays (RPAs) to monitor the effect of 9-cis-RA on the expression of death receptor, death ligands, IAP and TRAF family members in four different breast cancer cell lines. Breast cancer cells were treated for the indicated time with 9-cis-RA at a concentration of 10-6 M. (D) Western blot of whole cell extracts of 9-cis-RA-treated T47D cells and H3396 cells for 0, 12, 24, 48, 72 and 96 hours with anti-cIAP2. The nonspecific signal (n. sp.) confirms equal loading. As a positive control, breast cancer cells were treated with 50 μg/ml of hTNFα for 24 and 48 hours. (E) Reversibility of 9-cis-RA-induced cIAP2 gene expression. T47D cells were treated either in the absence or presence of 1 μM 9- cis-RA and after 3 days, total RNA was extracted. In parallel flasks, the medium was removed, and cells were washed and treated with either fresh control medium or medium with 10-6 M of 9-cis-RA and grown for additional 3, 6 and 9 days. Media and ligands were renewed every 3 days. RNA was isolated and analyzed by RPA as described in (A). Equal loading was confirmed by GAPDH RNA level. The images shown are from one representative experiment performed twice with similar results.