Fig. 2

NS-398 effects on apoptosis in LNCaP cells. Cells were cultured as in Fig. 1. At designated intervals adherent cells were harvested by gentle scraping and pooled with non-adherent cells floating in culture medium. Cells were collected by centrifugation then washed with PBS. (A) DNA laddering: Cells were collected following 72 h treatment with NS-398, then genomic DNA (20 µg) was separated on 1.5% agarose gels using TAE buffer. DNA was visualized in the SYBR-green stained gel and photographed. Gel is representative of data from three separate experiments. Lanes 1–2, C4-2b; lanes 3–4, LNCaP; lanes 1–3, DMSO controls; lanes 2–4, NS-398 treatment (10 uM) B). (B) Caspase-3 activity: Cells were collected following 48 h treatment with NS-398. 3 µg total cellular protein from cell lysates was used to determine protease activity toward the synthetic peptide Asp-Glu-Val-Asp (DEVD). Free pNA produced by DEVD cleavage was quantified by absorbance at 405 nm using an ELISA plate reader. Caspase-3 activity is expressed pNA optical density units/mg total cell lysate protein. Open, LNCaP, DMSO control; closed, LNCaP, NS-398. Caspase-3 activity could not be detected in C4-2b cells and extremely low basal levels detected in zero hour LNCaP cultures was attributed to basal cell lysate protease activity, as this activity was lost upon 1 mM protease inhibitor cocktail (Gibco/BRL) inclusion in the assay. Data are mean of three separate experiments performed in duplicate. Error bars = SE of means.