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. Author manuscript; available in PMC: 2010 Jun 24.
Published in final edited form as: Apoptosis. 2009 Jul;14(7):849–863. doi: 10.1007/s10495-009-0356-4

Fig. 2.

Fig. 2

4HPR functions as a prooxidant in PWR-1E and DU-145 cells. a PWR-1E cells were exposed for the indicated times to 1, 2.5, or 5 μM 4HPR or an equal volume of the vehicle Me2SO (control), and examined for the oxidation of 2′,7′-dichlorofluorescin to DCF using a microplate spectrofluorimeter. The assay described above was repeated using DU-145 cells exposed Me2SO (control), 5 μM 4HPR, 5 μM HQNO, or 4HPR and HQNO. The spectrofluorimeter preformed 12 fluorescence measurements per well and calculated the mean value. Triplicate wells for each sample were measured. b Immunoblot analysis of cellular proteins in DU-145 cells exposed for 24 h to Me2SO (control) or 5 μM 4HPR. The antibodies included mitochondrial aconitase (ACO2), BCL-XL, cytochrome c oxidase subunit II (COII), epidermal growth factor receptor (EGFR), Mn superoxide dismutase (MnSOD), and the loading control β-actin. The band intensity was normalized as a percent of the loading control β-actin using ImageJ software, and the percent decrease or increase of the examined proteins in the 4HPR-treated cells was measured relative to the same proteins in the control cells. c PWR-1E and DU-145 cells were pretreated for 2 h with 2.5 mM NAC followed by a 2.5 h exposure to 5 μM 4HPR or an equal volume of the vehicle Me2SO (control). The cells were examined for the oxidation of 2′,7′-dichlorofluorescin to DCF using a microplate spectrofluorimeter as described in a. ROS generation rates (fluorescence units/min, FU/min) were derived from the slopes of lines obtained between 30 and 120 min from triplicate wells in 6-well tissue culture plates. The results are expressed as the mean of triplicate samples for each treatment ± SD (error bars)(* P < 0.001 compared to control, ** P < 0.01 compared to 4HPR). d The PWR-1E and DU-145 cells were assessed for hypoploid cells by cytofluorometric analysis as described in Fig. 1a after a 2-h pretreatment with NAC followed by a 24-h (PWR-1E) or 48-h (DU-145) exposure to the vehicle Me2SO (control) or 4HPR. The results are expressed as the mean of triplicate samples for each treatment ± SD (error bars) (* P < 0.001 compared to control, ** P < 0.001 compared to 4HPR)