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. 2016 Jan 27;7(8):9135–9149. doi: 10.18632/oncotarget.7035

Figure 4. Lapatinib inhibits transcription of CIP2A.

Figure 4

A. Effect of lapatinib on CIP2A protein degradation. Cells were treated with 100 μg/ml pan-translation inhibitor cyclohexamide (CHX) in the presence (right) or absence (left) of lapatinib (5 μM) for the indicated period of time, then the stability of CIP2A protein in whole-cell lysates was assessed by western blot. The addition of lapatinib did not significantly affect CIP2A degradation. B. Lapatinib inhibited CIP2A transcription. Cells were treated with lapatinib at the indicated doses for 24 hours, after which total RNA was isolated and CIP2A mRNA was assayed by real-time quantitative PCR. Columns, mean (n = 3); bars, SD. C. Luciferase reporter assay of CIP2A proximal promoter regions upon lapatinib treatment. MDA-MB-468 cells were transfected by Firefly luciferase reporter vectors carrying CIP2A promoters of different lengths as indicated, and Renilla vectors for 24 hours and then treated with 5 μM lapatinib or DMSO for 24 hours. Cell lysates were then assayed for dual luciferase activity as described in Materials and Methods. Columns, mean (n = 3); bars, SD; *, P < 0.05. D. lapatinib disturbed binding of Elk1 to the CIP2A promoter region. Left, lapatinib decreased Elk1 translocation from the cytosol to the nuclei. Nuclear and cytoplasmic extracts were prepared from MDA-MB-468 cells treated with lapatinib (5 μM) or DMSO for 24 hours. Cell lysates were western blotted for Elk1, and Ets1. Lamin B and Tubulin were used as a loading control. Right, chromatin immunoprecipitation assays of the CIP2A promoter were performed as described in Materials and Methods. Soluble chromatin was immunoprecipitated with Elk1, Ets1 or IgG (negative control) antibodies. Immunoprecipitates were subjected to PCR with primer pairs specific to the CIP2A promoter (−16 to −139 bp). The gel shown is representative of three independent experiments. Anti-RNA polymerase II antibody and GAPDH primers were used as a positive control for the assay technique and reagent integrity. E. Ectopic expression of Elk1 with pCMV-Elk1-GFP vector upregulated CIP2A expression, and suppressed lapatinib-induced CIP2A inhibition.