FIG. 3.

A: Endogenous RAGE expression is involved in adipocyte hypertrophy. 3T3-L1 preadipocytes were cultured in DMEM with 10% FBS for 2 days. The cells were harvested, suspended, and transfected with predesigned siRNA for mouse RAGE using electroporation system. Twenty-four hours after siRNA transfection, the cells were infected with Ad-RAGE or Ad-LacZ. One day after adenoviral infection, differentiation was induced. Adipocyte differentiation was determined by Oil Red O staining 3 and 6 days after differentiation. Scale bars, 100 μm. *P < 0.05, Student t test. B: RAGE ligation with endogenous ligand(s) is indispensable for RAGE-stimulated adipocyte hypertrophy. The cells were transfected with siRNAs for control (cont), S100b alone, HMGB1 alone, or S100b + HMGB1, infected with Ad-LacZ or Ad-RAGE, and differentiated as described in A. In S100b and HMGB1 double-knockdown condition, addition of S100b (100 nmol/L), HMGB1 (10 ng/mL) alone, or both ligands during differentiation step negates the effect of siRNAs. Right panel shows the effect of RAGE overexpression or the effects of siRNAs for S100b and HMGB1 on mRNA levels of respective ligands. *P < 0.05 vs. control, **P < 0.05 vs. LacZ, Student t test. C: Effects of RAGE ligands on adipocyte hypertrophy in the presence or absence of RAGE overexpression. Indicated concentrations of HMGB1 or S100b were added during differentiation step following adenoviral infection as described in A. HPF, high-power field.