3DG Induces ER Stress Pathway Figure 10. Effect of neutralization of a1b1 integrin on 3 DG-collagen-induced ER stress pathway. A, Fibroblasts were pretreated with or without the blocking antibodies anti-b1 ITG, anti-a1 ITG, and anti-a5 ITG (10 mg/mL) for 30 min and cultured on native collagen, or 1 mM 3 DG- collagen, for 24 h and analyzed for the production of ROS. Fibroblasts were incubated with DCFH-DA for 30 min and the level of intracellular ROS was determined by measuring the fluorescence at 480 nm/530 nm. B, GADD153 localization in the nucleus was analyzed by immunofluorescence using a Cy3-conjugated secondary antibody. Images were taken at 406 magnification on an epi-fluorescence microscope and the MFI of ten nuclei was processed by ImageJ. Arrows indicate nuclei containing GADD153. Scale bar represents 10 mm. C, Western blot of GADD153 expression after neutralization of b1, a1, and a5 integrins. The bars correspond to the densitometric value of GADD153 after normalization for b-actin. D, Caspase-3 activity detected using the Caspase-3 Colorimetric Correlate Assay. All comparisons are made against collagen treated with PBS unless otherwise indicated. Data are mean6SD (n = 3), *P,0.001. doi:10.1371/journal.pone.0011093.g010 PLoS ONE | www.plosone.org 12 June 2010 | Volume 5 | Issue 6 | e11093