Figure 8.

(A) Sialidase activity in live parental DU145, PC3, and resistant DU145GemR and PC3GemR cells. Cells were allowed to adhere on 12 mm circular glass slides in media containing 10% fetal calf serum for 24 hours. After removing media, 0.318 mM 4-MUNANA substrate (2′(4-methlyumbelliferyl)-α-N-acetylneuraminic acid) in Tris-buffered saline pH 7.4 was added to live cells alone (control) or with EGF at 100 μg/mL. The substrate is hydrolyzed by sialidase to give free 4-methylumbelliferone, which has a fluorescence emission at 450 nm (blue color) following excitation at 365 nm. Fluorescent images were taken at 2 minutes after adding substrate by using epifluorescent microscopy (10× objective). (B) The mean fluorescence of 50 multipoint replicates was quantified by using the ImageJ software. Results were compared by one-way ANOVA at 95% confidence interval by using Bonferroni test. Data are a representation of one of two independent experiments showing similar results.

Abbreviations: EGF, epidermal growth factor; ns, not significant; ANOVA, analysis of variance.