The combination of cisplatin and irradiation increases HMGB1 protein secretion and cell surface expression of calreticulin protein in MB49 cells. (A) MB49 cells are treated with cisplatin at 0.6 mg/L and/or irradiated with a single fraction of 10 Gy. Medium is collected two and five days after treatment, and HMGB1 protein expression levels in each medium are examined in duplicate by ELISA (n = 3/group). (B) MB49 cells are treated with cisplatin at 0.6 mg/L and/or irradiated with a single fraction of 10 Gy. Cells are collected, and cell surface calreticulin protein expression levels are examined by flow cytometry (n = 3/group). (C,D) Seven days after inoculation MB49 cells in the left hindlimb, mice are treated with cisplatin at 3 mg/kg and/or irradiation with a single fraction of 10 Gy. Tumor sections before and after CRT (n = 3/group) are stained for HMGB1 (C) and calreticulin (D). Left, representative images; right, comparison of the mean (± standard error of the mean [SEM]) number of HMGB1- or calreticulin-positive cells counted in five fields of view (FOV). All data are shown as the mean ± SEM. Asterisks indicate p values comparing two groups, as indicated in the figure. NS, not significant; * p < 0.05; ** p < 0.01.