FIG 4.

(A) Effect of 4EGI-1 and etoposide on cellular apoptosis in LNCaP cells. Subconfluent LNCaP cells were treated for 24 h with either 10 μM etoposide, 50 μM 4EGI-1, or both. Both floating and attached cells were collected and lysed following treatment. Equal amounts of protein were subjected to SDS-PAGE and transferred to a PVDF membrane. PARP, cleaved PARP, and β-actin were detected. (B) 4EGI-1 does not stimulate p53 phosphorylation at serine 15 in LNCaP cells. Subconfluent LNCaP cells were treated with either 10 μM etoposide, 50 μM 4EGI-1, or both for 4 h. After treatment, the cells were lysed. Phosphor-p53 at Ser15 and β-actin were detected following SDS-PAGE and Western blotting. (C) 4EGI-1 does not cause DNA double-strand breaks in LNCaP cells. Subconfluent LNCaP cells grown on chamber slides were treated with 10 μM etoposide or 50 μM 4EGI-1 for 4 h. After treatment, indirect immunofluorescence was performed with phosphor-histone H2A.X antibody as described in Materials and Methods, followed by DAPI staining. Images were taken using a confocal microscope at ×60 magnification.