Figure 4

Bortezomib downregulates transcription of CIP2A. A, After cells were treated with 100 μg/ml translation inhibitor cyclohexamide (CHX) in the presence or absence of 500 nM bortezomib for the indicated length of time, the stability of CIP2A protein in whole-cell lysates were assessed by western blot. In bortezomib-sensitive cells (HCC 1937, MDA-MB-231, and MDA-MB-468), the addition of bortezomib did not affect CIP2A degradation. B, Bortezomib inhibits CIP2A mRNA in a time-dependent manner. Cells were treated with bortezomib at 500 nM for the indicated length of time, after which total RNA was isolated and CIP2A mRNA were semi-quantified using PCR as described in Methods. Columns, mean (n = 3); bars, SD. C, Bortezomib inhibits CIP2A mRNA in a dose-dependent manner in sensitive cells (left), but not in resistant cells (right). Cells were treated with bortezomib at the indicated doses for 36 hour, after which total RNA was isolated and CIP2A mRNA was analyzed by real-time quantitative PCR (qRT-PCR). Columns, mean (n = 3); bars, SD. CIP2A, cancerous inhibitor of PP2A; PCR, polymerase chain reaction; SD, standard deviation.