Anti crt

The PanC02 cells were cultured on a round coverslip or in 12-well plates. After different treatments (PBS, PBS + US, PPIX NDs, PPIX NDs + US, PMPS NDs, PMPS NDs + US), the cell was fixed using 4% paraformaldehyde at Day 2. The expression of HMGB-1 and CRT was evaluated by Western blot and immunofluorescence using anti-HMGB-1 (Abcam) and anti-CRT (Abcam). Moreover, the expression of CRT was also evaluated by FCM.

The murine colon carcinoma cell line, CT26, murine mammary carcinoma cell line, 4T1 (gift of Yang-Xin Fu), and three human colon cancer cell lines HCT116, SW480, and HT29 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) where each lot was STR profiled. All cell lines were authenticated either by ATCC or IDEXX (4T1), tested for mycoplasma, and used at low passage numbers within 6 months. Cells were grown in the following media: RPMI-1640 (CT26); McCoy's 5A (HCT116); minimum essential medium Eagle (SW480); and Dulbecco's modified Eagle's mMedium (HT29). All media were supplemented with 10% FCS, 2 mM glutamine, and penicillin–streptomycin antibiotic, and the cells were incubated at 37 °C in 5% CO₂. RB solution of 10% was provided by Provectus Biopharmaceuticals (Knoxville, TN, USA). Annexin V-Biotin Apoptosis Detection Kit, streptavidin-FITC, and streptavidin-APC were purchased from eBioscience (San Diego, CA, USA). Anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CRT (Abcam, Cambridge, MA, USA), anti-HSP90, and anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA) antibodies were purchased and used according to the manufacturer's specifications. DAPI, quinacrin dihydrochloride, and chloroquine diphosphate were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Phagocytosis of autophagic and apoptotic HeLa cells was performed using human macrophages as phagocytes. Human macrophages were incubated with autophagic and apoptotic HeLa cells in a ratio of 10∶1 (10 dead cells per phagocyte) for 4 h at 37°C. Dead cells were stained with 1 mg/mL Hoechst 33342 for 5 min at 37°C before being added to the macrophage cultures. In inhibition experiments, chicken polyclonal anti-CRT (abcam, Cambridge, UK) added 30 minutes before phagocytosis assay was used. The phagocytosis rate (number of macrophages engulfing at least one autophagic or apoptotic cell) and phagocytosis index (number of autophagic or apoptotic cells internalized per macrophage) were reported by scoring at least 500 cells for each experiment, under a fluorescence microscope NIKON Eclipse 80i (Nikon, Tokyo, Japan), with Plan Fluor objectives (Nikon). The percentage of cells binding but not ingesting autophagic or apoptotic cells was calculated and expressed as percentage of binding.