Induction of osteopontin expression by PM is oxidative stress-dependent. A, ascorbate pre-treatment (200 μM for 5 h) prior to a 24-hour exposure to 12, 24, or 48 μg/ml PM reduced osteopontin secretion compared to cells exposed to PM alone. Data are presented as mean ± S.D. osteopontin concentrations, as detected by electrochemiluminescence. N = 6 in each case. B, similar to the secretion studies, immunocytochemistry showed that ascorbate pre-treatment blunted the increase in osteopontin immunostaining caused by PM exposure. Graphical data are mean pixel intensities from 10 cells at each concentration. In A and B, *p < 0.05 compared to controls and #p < 0.05 compared to PM alone. C, Western blotting studies detected the presence of bands at 66 kDa and 33 kDa representing full-length and MMP-3 cleaved osteopontin, respectively. Ascorbate pre-treatment caused a reduction in the PM-induced elevation of osteopontin protein levels, which was most prominent in the MMP-3 cleaved form. Lane one, DMSO; lane two, ascorbate alone; lane three, PM; and lane four, PM with ascorbate pre-treatment. Graphical data are band densities obtained by densitometric analysis of Western blots.