Research Paper Volume 13, Issue 6 pp 8380—8395

Figure 2. Extracellular HMGB1 promotes the sphere formation, invasion and EMT process in CD44-dependent way. (A) Immunoblot analysis shows rhHMGB1 promotes CD44 expression in a dose-dependent manner. HepG2 and HCCLM3 cells were cultured with different concentration of rhHMGB1 for 24h. (B) Q-PCR analysis shows that CD44 is upregulated by rhHMGB1 in a dose-dependent manner. (C, D) rhHMGB1 treatment promotes HCC sphere formations in CD44-dependent way. Number and size of spheres were photographed and analyzed. HepG2 and HCCLM3 cells were transfected with negative siRNA or CD44 siRNA and then cultured with rhHMGB1 (1μg/ml) for 24h. (E, F) Invasion experiments show rhHMGB1 promotes invasive abilities of HCC cells in CD44-dependent way. The invaded cells were counted and results were analyzed. HepG2 and HCCLM3 cells were transfected with negative siRNA or CD44 siRNA and then cultured with rhHMGB1 (1μg/ml) for 24h. (G) Immunoblot analysis shows that targeting CD44 inhibits EMT process caused by rhHMGB1. HepG2 and HCCLM3 cells were transfected with negative siRNA or CD44 siRNA and then cultured with rhHMGB1 (1μg/ml) for 24h. Data are means ± SEM, * means p<0.05, ** means p<0.01, *** means p<0.001 by unpaired student T test.