3DG Induces ER Stress Pathway Figure 4. 3DG-collagen increases expression of Nox4 in the dermal fibroblast. Fibroblasts were cultured on either native collagen or 1 mM 3DG-collagen and treated with or without 5 mM AG or 40 mM meglumine for 24 h. A, Nox4, Nox1, and Nox2 mRNA expression levels were quantified by real-time PCR. All transcripts were normalized to b-actin. B, Expression levels of Nox4 were analyzed by Western blot and b-actin served as a loading control. Results were quantified by densitometric scanning of the Western blot and normalized for b-actin. C, Localization of Nox4 in fibroblasts treated the same as in A and B was analyzed by immunofluorescence with the anti-Nox4 polyclonal antibody and Cy2-conjugated secondary antibody. Images were taken at 406 magnification on an epi-fluorescence microscope. Scale bar represents 10 mm. D, Inhibition of Nox4 reduces the level of intracellular ROS. Fibroblasts were pretreated for 1 h with either vehicle, DMSO or NOX inhibitor, apocynin (1 mM) and cultured on native collagen, 1 mM 3DG-collagen, or cultured on native collagen and treated with 50 mM H2O2 for 24 h. Fibroblasts were then incubated with DCFH-DA for 30 min and the level of intracellular ROS was determined by measuring the fluorescence at 480 nm/530 nm. Comparisons are made against collagen treated with DMSO and/or PBS unless otherwise indicated. Data are mean6SD (n = 3), **P,0.001, *P,0.02. doi:10.1371/journal.pone.0011093.g004 PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e11093