activation of Nox4. Furthermore, apocynin was found to only partially inhibit the level of ROS in fibroblasts cultured on native collagen with H2O2 suggesting that apocynin is inhibiting the ROS induced by the Nox4 complex rather than affecting the induction of ROS by exogenous H2O2 (Figure 4D, p,0.02). 3DG-collagen-induced phosphorylation of p38 MAPK is dependent on upstream ROS During times of ER stress, ROS have been shown to activate the stress kinase p38 MAPK [35,37,44]. To determine if 3DG- collagen-induced ROS are responsible for increased phosphory- lation of p38 MAPK, fibroblasts were pretreated with the antioxidant ascorbic acid and the Nox inhibitor apocynin, and cultured on either native collagen, 3DG-collagen, or native collagen and treated with H2O2 and protein levels were measured by Western blotting. Fibroblasts cultured on 3DG-collagen increased the phosphorylation of p38 MAPK to 175%64.1%. As a positive control for ROS-induced p38 MAPK activation, fibroblasts cultured on native collagen and treated with H2O2 increased the phosphorylated p38 MAPK to 181%63.3%. Pretreatment with ascorbic acid reduced the phosphorylation of p38 MAPK in fibroblasts cultured on 3DG-collagen or treated with H2O2 to that seen in fibroblasts cultured on native collagen (Figure 5, p,0.0001). Additionally pretreatment with apocynin reduced the level of phosphorylated p38 MAPK in fibroblasts cultured on 3DG-collagen, but not in fibroblasts treated with H2O2 (Figure 5, p,0.0001). These results suggest that 3DG- 3DG Induces ER Stress Pathway collagen-induced p38 MAPK is dependent on upstream produc- tion of ROS by Nox4. 3DG-collagen-induced GADD153 expression is dependent on upstream ROS and p38 MAPK activation 3DG-collagen-induced ROS can lead to phosphorylation of p38 MAPK, which is essential for the activation of GADD153; therefore, the functional role of ROS and p38 MAPK in GADD153 induction was assessed [35,37,43,44]. To determine whether GADD153 induction by 3DG-collagen was a result of free radical-mediated effects, fibroblasts were pretreated with ascorbic acid or apocynin and then cultured on native collagen or 3DG-collagen for 24 h. Fibroblasts cultured on native collagen and treated with H2O2 were used as a positive control for ROS- induced GADD153 activation. The trafficking of GADD153 from the cytosol to the nucleus was found to be downregulated to 14.6 MFI62.1 and 16.3 MFI60.98 in response to ascorbic acid in fibroblasts cultured on 3DG-collagen or native collagen treated with H2O2, respectively (Figure 6A, p,0.007). The expression of GADD153 in the nucleus of fibroblasts pretreated with apocynin and cultured on 3DG-collagen was also reduced to 13.9 MFI61.2 (Figure 6A, p,0.007). Western blot was performed to verify the expression of GADD153. GADD153 expression was decreased in response to ascorbic acid and apocynin in fibroblasts cultured on 3DG-collagen, while only ascorbic acid reduced the level of GADD153 in fibroblasts cultured on native collagen treated with H2O2 (Figure 6B; 81%62.4% 3DG-collagen treated with ascorbic Figure 5. Phosphorylation of p38 MAPK is dependent on 3DG-collagen-induced ROS. Fibroblasts were pretreated for 1 h with 100 mg/mL of ascorbic acid, 1 mM apocynin, or DMSO and cultured on either native collagen, 1 mM 3DG-collagen, or treated with 50 mM H2O2 for 24 h. Whole cell lysates were extracted and Western blot analysis of p-p38 MAPK was performed. Total p38 MAPK was used as a loading control. The bars correspond to the densitometric value of p-p38 MAP kinase after normalization for total p38 MAP kinase. Data are mean6SD (n = 3), *P,0.0001. doi:10.1371/journal.pone.0011093.g005 PLoS ONE | www.plosone.org 6 June 2010 | Volume 5 | Issue 6 | e11093