Effect of SELN on apoptosis.

<p>(A) SOJ-6 and MiaPaCa-2 cells were starved then treated for 24h with SELN (16 nmoles cholesterol/ml) and lysed. Cell lysate proteins were separated on SDS-PAGE (80 µg of proteins/lane) and electrotransferred onto nitrocellulose membrane. The levels of Bax, Bcl-2 and β-actin were determined by probing membranes with specific antibodies as indicated. Western blots are representative of three independent experiments. Lane 1; control performed in the absence of SELN, Lane 2; in the presence of SELN 3.0, Lane 3; in the presence of SELN 6.0. The right panels in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047480#pone-0047480-g004" target="_blank">figure 4A</a> represents the quantification of western blots and the ratio Bax/Bcl-2. Data are means (± SD) of three independent experiments using the NIH Image program (Mann-Whitney test). (B) Apoptosis was determined using the Terminal Transferase dUTP Nick End Labeling (TUNEL). SOJ-6 cells were starved then treated with SELN6.0 (16 nmoles cholesterol/ml) for 24h then fragmented DNA was stained according to the protocol given with the ApopTag® Red In Situ Apoptosis Detection Kit (right pictures). Nuclei were counterstained with DAPI (left pictures). Apoptotic cells were visualized under a Zeiss fluorescence microscope equipped with a digital camera. The ratio of apoptotic cells to total cells was counted in 5-to-10 random area, in both control and SELN6.0 treated SOJ-6 cells (right histogram, (means (± SD) of three independent experiments, Mann-Whitney test), scale bar = 100 µm. (C) SOJ-6 cells were incubated 4 h with medium (control), with SELN6.0 (16 nmoles of cholesterol/ml, column 2), with SELN6.0 and Z-IETD-fmk (a caspase-8 inhibitor, 10 µM, column 3) or Z-LEHD-fmk (a caspase-9 inhibitor, 10 µM, column 4). After adding freshly prepared SELN6.0, cells were incubated for another 24h. After washing and fixation, cleaved caspase-positive cells were counted under fluorescent microscopy. Results are means (± SD) of fluorescent cell amounts collected in 10 fields per assay (at least 3 independent assays). Results are expressed as percentage of cleaved caspase-positive cells relatively to controls (Mann-Whitney test).