Pd l1

Protein was extracted from compound-treated cells. Protein was separated by electrophoresis of the sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and transferred to the polyvinylidene fluoride membrane (PVDF). The membranes were incubated overnight at 4 °C with primary antibodies after blocking in 5% BSA/TBST buffer for 2 h. The following primary antibodies were used at 1:1000: PD-L1 (ABCAM, cat. ab213524), AKT (CST, cat. 4691 T), p-AKT (beyotime, cat. AF1546), mTOR (CST, cat. 2972S), p-mTOR (CST, cat. 2971S), Bcl-2 (HuaBio, cat. HN0921), Caspase 3 (CST, cat. 9662S), c-Caspase 3 (beyotime, cat. AC033), PARP (CST, cat. 9542 T), c-PARP (CST, cat. 9541 T), GAPDH (beyotime, cat. AF1186), and β-actin (CST, cat. 4970 T).

On Day 10, after the beginning of treatment, the peripheral blood was collected for detection of TNF-α, IL-6, IFN-γ, and IL-10 levels in plasma using ELISA kits (Abclonal). The primary tumors were isolated from the treated mice and cut into sections for further examination. These sections were separately processed with primary antibodies against CRT, HSP 70, and PD-L1 (Beyotime) according to the manufacturer's protocols and then stained with secondary antibodies, followed by microscopic observation.

4T1 cancer cells (1 × 10⁵) seeded in confocal petri dish were incubated with PM, PM@RM and PM@RM-T7 (at the equivalent mPDA = 200 μg/mL, Met = 60 μg/mL) for 6 h. Afterward, the cells were laser irradiated (808 nm, 1.0 W/cm², 5 min) or not. The medium was changed to a fresh medium. After another incubation for 24 h, 4T1 cells were washed with PBS (pH 7.4) and incubated with 5% bovine serum albumin for 30 min to block unspecific binding of antibodies. Then, the cells were respectively incubated with anti-calreticulin (CRT) and anti-heat shock protein (HSP70) antibodies (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C, followed by incubation with the corresponding secondary antibodies for 2 h at 37 °C in the dark. Finally, the cells were visualized under a CLSM (Zeiss) system. The procedures of immunofluorescence (IF) staining of PD-L1 (Beyotime) were the same as the above.