Fig. 1. Dinaciclib sensitizes HER2-amplified breast cancer cells to lapatinib and liberates BAK from MCL-1.

A BT-474 and MDA-MB-453 cells were treated with no drug, 1 μM lapatinib, 100 nM dinaciclib, their combination and the combination of 1 μM lapatinib with 10 μM A1210477 for 6 and 12 h, respectively. Whole-cell lysates were prepared, subjected to western blotting and probed for the indicated proteins. B Cells from SKBR3, BT-474, MDA-MB-453, and HCC-1419 HER2-amplified breast cancer cell lines were treated with no drug or 100 nM dinaciclib for 2 h, and levels of the abundance of MCL-1 mRNA were analyzed by qPCR. Data are normalized to ACTB; n = 3; error bars indicate ±SEM. C BT-474 and MDA-MB-453 cells were treated with increasing concentrations of lapatinib and 10 μM A1210477 or with increasing concentrations of lapatinib and 100 nM dinaciclib for 24 and 72 h respectively, and the percentage of viable cells was determined. n = 3; error bars indicate ±SD. D BT-474 and MDA-MB-453 cells were treated with no drug or the combination of 1 μM lapatinib and 100 nM dinaciclib for 24 and 72 h, respectively and the percentage of annexin V/PI-positive cells was determined by FACS. n = 3, error bars indicate ±SD (“No Rx”: No drug). E MCL-1 complexes were immunoprecipitated from the indicated HER2-amplified breast cancer cell lines following 6 h (BT-474) and 12 h treatment (MDA-MB-453) with no drug, 1 μM lapatinib, 100 nM dinaciclib, and their combination. An IgG-matched isotype antibody was served as an immunoprecipitation control. The interaction between MCL-1 and BIM EL/BAK proteins was investigated (“No Rx”: No drug). For Fig. 1B–D two-tailed Student’s t test was performed. p values were corrected for multiple testing using the Bonferroni method. Differences were considered statistically different if p < 0.05. A p value < 0.05 is indicated by *, p < 0.01 by **, p < 0.001 by ***, p < 0.0001 by ****.