3DG Induces ER Stress Pathway Figure 6. Inhibition of ROS, and p38 MAPK abrogates GADD153 expression in fibroblasts cultured on 3DG-collagen. Fibroblasts were pretreated with 100 mg/mL of ascorbic acid, 1 mM apocynin, or 10 mM SB202190 for 1 h, and cultured on native collagen, 1 mM 3DG-collagen or treated with 50 mM H2O2 for 24 h. A, Fibroblasts were stained and analyzed for the expression of GADD153 in the nucleus by immunofluorescence using a Cy3-conjugated secondary antibody. Representative images were taken at 406 magnification on an epi-fluorescence microscope, and the MFI of ten nuclei was analyzed by Image J. Bars correspond to the MFI of treated fibroblasts. Arrows indicate nuclei containing GADD153. Scale bar represents 10 mm. B, Western blot for the expression of GADD153 in whole cell lysates. b-actin was used as a loading control. The bars correspond to the densitometric value of GADD153 after normalization for b-actin. All comparisons are made against collagen treated with PBS unless otherwise indicated. Data are mean6SD (n = 3), ***P,0.0001, **P,0.0005, *P,0.007. doi:10.1371/journal.pone.0011093.g006 PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e11093