After treated with MS NPs for 48 h, MB231 cells were washed with cold PBS and lysed in lysis buffer (1× phosphatase inhibitor cocktail, and RIPA) on ice bath. Then cells lysates were collected with a cells-scraper and centrifuged (14,000 rpm at 4 °C for 15 min), and the supernatant was collected for protein quantitation. Proteins were separated by 10% SDS-PAGE and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, USA). The membrane was blocked with 5% BSA in TBST. For identifying PD-L1, the membrane was incubated with primary antibody for PD-L1 (Cells Signaling Technology) or GAPDH (Cells Signaling Technology) overnight at 4 °C. After being washed with TBST, the membrane was incubated with HRP-conjugated anti-rabbit secondary antibody, and reacted with chemiluminescent substrate. Then reactive bands were obtained using a ChemiDocTM Touch gel imaging system (Bio-Rad, USA). As for densitometric analysis, all PD-L1 bands were normalized to the internal reference GAPDH, respectively. Image Lab software (Bio-Rad, USA) was used for densitometric analysis.

Post-treatment, MB231 cells were collected and blocked with 10% normal goat serum at 4 °C for 1h. Then MB231 cells suspension was labelled with anti-PD-L1 antibody (Alexa Flor® 647, Abcam) at 4 °C for 30 min. After thoroughly washed with washing buffer (cold PBS containing10% normal goat serum), MB231 cells were measured with a Canto II flow cytometry (BD Bioscience, CA) equipped with a 633 nm laser. FlowJo software was used to analyze the data.

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