4T1 cells (4 × 105 cells per well) were seeded in six-well plates and cultured for 24 hours. Then, cells were incubated with free PR104A or free CPT (10 μM) for 12 hours (n = 3). After incubation, the cells were washed, collected, and broken by freezing and thawing. The free drug ratio was determined by ultrafiltration.

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