Figure 7

Over-expression of the super-repressor of NF-κB activation, IκBα-SR(S32A/S36A), leads to significant abrogation of retinoic acid-mediated inhibition of etoposide-induced apoptosis. (A) T47D-vector and T47D-IκBαSR cells were untreated or incubated for the indicated time with hTNFα (50 ng/ml). Extracts were analyzed by western blotting with an IκBα antibody. Equal loading was confirmed with an anti-β-actin antibody. The images shown are from one representative experiment performed three times with similar results. (B) T47D-vector or T47D-IκBαSR cells were untreated or treated with 9-cis-RA for 48 h and expression of cIAP2 and β-actin were analysed by Reverse-Transcriptase Polymerase Reaction and real time PCR. The values represent the mean ± SD of three different experiments performed in duplicate. (C) T47D-vector or T47D-IκBαSR cells were pretreated with or without 9-cis-RA for 30 h, followed by treatment with etoposide 100 μM as previously indicated. At the indicated times, cell lysates were analyzed by western blot for the expression of cleaved caspase-3, cIAP2 and β-actin using specific antibodies. The images shown are from one representative experiment performed three times with similar results. (D) T47D-vector or T47D-IκBαSR cells were pretreated with or without 9-cis-RA for 30 h, followed by treatment with etoposide 100 μM for 72 h. The percentage of apoptotic cells was determined by FACS analysis after staining with propidium iodide. The values represent the mean ± SD of three independent experiments performed in duplicate. Asterisks denote the existence of statistically significant differences between the indicated groups; N.S.: not significant (Student's t-test).