Effect of D609 on HER2 expression in SKBr3 cells continuously exposed to trastuzumab. (a) CLSM analyses of cells fixed after exposure for the indicated time intervals to trastuzumab, in the presence or absence of D609. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. (b) Decrease in the overall HER2 content, detected by Western blotting, in total lysates of cells after incubation for different time intervals with D609 (50 μg/mL), trastuzumab (HERC, 10 μg/mL), or their combination (COMB). CTR, untreated control cells. Both CLSM and immunoblotting analyses were repeated 3 times.