HMGB1 was released from dying cells in vitro. (a, b) Western blot and qRT-PCR analysis of the baseline protein and mRNA levels of HMGB1 in pancreatic cancer cell lines (SW1990, Panc1, and AsPC1). GAPDH expression was detected as loading control. (c) Western blot analysis of the protein expression levels of HMGB1 in pancreatic cancer cells lines (SW1990, Panc1, and AsPC1) at 24 h post-irradiation (0, 4, 8, or 10 Gy). GAPDH was detected as a loading control. (d) Western blot analysis of HMGB1 protein-expression levels in irradiated (8 Gy) cancer cells (SW1990, Panc1, and AsPC1) at 0, 6, 12, 24, 36, 48, or 72 h post-treatment. GAPDH was detected as a loading control. (e) ELISA-based analysis of the HMGB1concentration in the culture supernatants of 8-Gy-irradiated cancer cells (SW1990, Panc1, and AsPC1) at 0, 6, 12, 24, 36, 48, 72 h post-treatment. (f) Immunofluorescence assays were performed to access the expression level and location of HMGB1 in pancreatic cancer cell lines (SW1990, Panc1, and AsPC1) at 48 h post-irradiation (8 or 12 Gy). Red: HMGB1; Blue: nucleus. Scale bar, 50 μm. Experiments were repeated three times, and the data are expressed as the mean ± SEM. *P < .05, **P < .01, ***P < .001. Statistical analysis was performed using Student's t-test (b, c right).