To determine the expression levels of the apoptotic protein of Caspase-3 and the ferroptosis-related proteins of AIF and EndoG, 4T1 cells were seeded into six-well plates at a density of 105 U/cm2 and incubated overnight at 37°C under an atmospheric CO2 level of 5% until the cell confluence reached around 70%. Subsequently, the culture media were replaced by fresh ones containing TCPS (blank control), ACC-CaSi-PAMAM-FA/mPEG, DOX, ACC@DOX-CaSi-PAMAM-FA/mPEG, ACC@DOX. Fe2+-CaSi-PAMAM-FA/mPEG, and MMP-2–treated ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG and further incubated for 24 hours. The concentration for all nanoparticles was 34 μg/ml, and the DOX concentration was 1 μg/ml, which is to ensure the comparability of the results. The cells were then lysed with Laemmli Sample Buffer (Bio-Rad), and the total protein was quantified by electrophoresis using a BCA protein kit (Beyotime) and 12% SDS–polyacrylamide gel electrophoresis. The proteins were then transferred from the gel onto polyvinylidene difluoride membrane (Immobilon P, Millipore) and blocked by primary and secondary antibodies. The images were captured on a molecular imager Versa Doc MP 4000 system (Bio-Rad).

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