Fig. 3.

Dying cell-derived HMGB1 regulated CD133⁺ cancer cells stemness. (a) Western blot analysis of shRNA-mediated knockdown of HMGB1 protein expression in pancreatic cancer cell lines. GAPDH expression was detected as a loading control. (b) ELISA-based analysis of HMGB1 concentrations in the culture supernatant of HMGB1-knockdown cancer cell. (c, d) The number and size of spheres formed from CD133⁺ cancer cells were measured following a 14-d co-cultured with the following agents: i) lethally irradiated HMGB1⁺ cancer cells; ii) HMGB1⁻ cancer cells; iii) shControl + rhHMGB1(150 ng/mL); iv) shHMGB1 + rhHMGB1(150 ng/mL); v) various concentrations of rhHMGB1(50, 100, 150, and 200 ng/mL). (e, f) Western blot and qRT-PCR analysis of mRNA- and protein- expression levels of stem cell markers (Oct4, Sox2, and Nanog) in CD133⁺ cancer cells after a 14-d co-cultured with the following agents: i) lethally irradiated HMGB1⁺ cancer cells; ii) HMGB1⁻ cancer cells; iii) rhHMGB1 (150 ng/mL). GAPDH was detected as a loading control. Scale bar, 1000 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 (a right, b, d, e, f right).