To evaluate the cellular uptake of Dox conjugates, MMTV-PyMT cells were seeded in a 96-well high-content imaging plate (Corning) at 5000 cells per well and incubated overnight. Cells were washed with PBS and treated with free Dox, Dox-SA, or Dox-CBD-SA dissolved in DMEM [sodium pyruvate (110 mg/liter), 10% heat-inactivated FBS, 1% penicillin-streptomycin, and Phenol red (−)] at a concentration of 50 μM equivalent of Dox. After incubation, cells were washed twice, treated with 75 nM LysoTracker Deep Red, and further incubated for 30 min at 37°C. Cells were washed twice and observed by an IX83 microscope (Olympus) with ×60 magnification. Images were processed using ImageJ software (National Institutes of Health, NIH). Scale bar, 20 μm.

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