Figure 4.

SPRED2 affects the expression of stemness markers. (A) Cell extracts were prepared, and the presence of each protein was evaluated by western blotting. Band densities were digitized and semi-quantitated (n = 3, each). (B) HepG2 cells were treated with 20 μM PD98059 or vehicle (DMSO) for 24 h, cell lysates were prepared, and the presence of each protein was evaluated by western blotting. Band densities were digitized and semi-quantitated (n = 3, each). (C) Cells were stained with Alexa Fluor 488-conjugated anti-human CD44 antibody or anti-human CD90 antibody, and the percentage of positive cells was analyzed by flowcytometry (n = 3, each). (D) HepG2 cells were treated with 20 μM PD98059 or vehicle (DMSO) for 24 h in standard culture conditions and stained with Alexa Fluor 488-conjugated anti-human CD44 antibody or anti-human CD90 antibody, and the percentage of CD44 or CD90 positive cells was analyzed by flowcytometry (n = 3, each). (E) CD44⁺ and CD90⁺ cells were isolated from HepG2 cells by using CD44 and CD90 microbeads. Cell lysates were prepared, and the presence of each protein was evaluated by western blotting. Band densities were digitized and semi-quantitated (n = 3, each). (F,G) CD44⁻CD90⁻ and CD44⁺CD90⁺ cells were isolated from HepG2 cells by using CD44 and CD90 microbeads. (F) Representative cell morphology is shown. Scale bars: 50 μm. (G) Cell lysates were prepared, and the presence of each protein was evaluated by western blotting. Band densities were digitized and semi-quantitated (n = 3, each). ^# p < 0.05, ^§ p < 0.01, two-tailed unpaired t test.