Figure 2

PI3K and P38 mitogen activated protein kinase (MAPK) but not SMAD3 contribute to the induction of Secreted protein acidic and rich in cysteine (SPARC) by transforming growth factor (TGF)-β. (A) HFL-1 cells were transfected with non-targeting control or SMAD3 siRNA for 24 h, starved of serum for 24 h, and then cell lysates were subjected to western blotting analysis for SMAD3 expression. Bar graph shows densitometric analysis of western blotting. (B) HFL-1 cells were transfected with non-targeting control or SMAD3 siRNA for 24 h, starved of serum for 24 h, and then stimulated with TGF-β (1 ng/ml) for 24 h. SPARC and PAI-1 gene expression were analyzed by real-time PCR, and normalized relative to 18S rRNA. Data are expressed as means ± SE of three independent experiments. **P < 0.01 versus non-targeting control. (C and D) After 24 h of serum starvation, HFL-1 cells were stimulated with TGF-β (1 ng/ml) for 24 h in the presence/absence of the inhibitors U0126 (MEK inhibitor), LY294002, PI103 (PI3K inhibitor), SB202190, SB239063 (p38 MAPK inhibitor), or SP600125 (JNK inhibitor). SPARC gene expression was analyzed by real-time PCR, and normalized relative to 18S rRNA. Data are expressed as means ± SE of three independent experiments. *P <0.05, **P <0.01 versus control. MEK, mitogen-activated protein kinase kinase; JNK, c-Jun N-terminal kinase.