Fig. 4

PI3K/Akt pathway and EMT program was responsible for the dying cell derived HMGB1 mediating metastasis. a Western blot analyzing the expression of PI3K-p85α, p-Akt, N-cadherin, vimentin, and E-cadherin in Panc-1 cells treated with PBS, N-S, HMGB1^−/−-S, N-S + EP, and rhHMGB1 (150 ng/mL). β-Tubulin was a loading control. b RT-PCR analyzing the expression of EMT-related transcriptional factors Zeb1, Slug, and Snail in Panc-1 cells treated with PBS, N-S, HMGB1^−/−-S, N-S + EP, and rhHMGB1 (150 ng/mL). GAPDH was a loading control. c Western blot showing the shRNA-knockdown efficiency of Zeb1. β-Tubulin was a loading control. d The invasion ability of Panc-1 cells following Zeb1 knockdown or treated with DMSO: MK2206 (AKT phosphorylation inhibitor) in the presence of rhHMGB1 (150 ng/mL) was detected by transwell assay. Magnification: × 20. Experiments were repeated three times and the data were expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. e Flow cytometry analyzing the percentage of CD133 cancer cells in Panc-1 cells treated with PBS, N-S, HMGB1^−/−-S, N-S+EP, and rhHMGB1 (150ng/mL)