Arsenite and UVA induce cellular ROS. HaCaT cells grown on glass coverslips to 50% confluence were treated with 3 μM arsenite (As), 10kJ/m2 UVA (UVA) or left untreated (NT). Dually treated samples were pre-incubated with 3 μM arsenite (As) for 24 hrs and then exposed to UVA. A superoxide-specific dye (5 μM DHE) was added 30 min. prior to fixation. Samples were rinsed with PBS, fixed using 3.7% para-formaldehyde, mounted on glass slide and observed with fluorescence microscopy 4 hours post UV exposure. Lower panels were treated as described above with the addition of the cell permeable SOD mimic MnTMPyP (sod,5 μM) 30 min. prior to exposures to demonstrate fluorescence is derived from superoxide (A). ROS quantification was carried out as described in “Experimental Procedures.” Each image is representative of multiple observations, and error bars indicate ± S.D. (B). ROS generation was monitored over time and quantified as described in “Experimental Procedures” for untreated or dark (), arsenite (■), UVA (▲) and dually treated (●) samples (C). All p values were <0.05 versus the untreated control. * = significantly different from untreated controls (NT), p<0.05; # = significantly different from singly treated samples, p<0.05. DHE, dihydroethidium.