Fig. 3

Dying-cell-derived HMGB1 regulates tumor-cell metastasis through TLR2 in vitro. a Western blot showing shRNA-knockdown efficiency HMGB1 in Panc-1 cancer cells. β-Tubulin was a loading control. b Panc-1 cells’ migration ability following being treated with N-S (supernatants from the parental cancer cells following 12 Gy radiation), HMGB1^−/−-S (supernatants from the HMGBl knockdown cancer cells following 12 Gy radiation), N-S + EP (HMGB1 inhibitor), and different concentrations of rhHMGB1 (50, 100, 150, and 200 ng/mL) for 12 h was analyzed by transwell invasion assays. Migratory cells were counted in at least three to four randomly-selected microscopic fields and the results are expressed as the mean ± SEM of migratory cells per microscopic field. Magnification: × 20. c Western blot showing shRNA-knockdown efficiency of RAGE, TLR2, TLR4, and CD24 in Panc-1 cells, respectively. β-Tubulin was a loading control. d The invasion ability of Panc-1 cells following knockdown: the receptor, treated with PBS, N-S, rhHMGB1 (150 ng/mL), was accessed by transwell assay. TLR2, rather than other receptors, mediated the HMGB1-induced Panc-1 cells’ metastasis. Magnification: × 20. Experiments were repeated three times and the data were expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001