Fig. 5. Che-1 counteracts NRAGE induced apoptosis. (A) P19 cells were transfected with siRNA targeting Che-1 (iChe-1) or with a non-specific siRNA control pool (Smart). After 24 hours, apoptosis was induced with 1 µM retinoic acid (RA). Cell-death was measured by Trypan Blue exclusion after 48 hours. Transfected cells were subjected to western blot analysis to verify reduced Che-1 expression levels after siRNA treatment (top). Note that cells with low Che-1 levels are more responsive to RA treatment. (B) P19 cells were transfected with Myc-tag expression vectors for NRAGE, Che-1 and Che-1-C proteins. After 48 hours, cell death was measured by Trypan Blue exclusion. Each point represents the mean of three independent experiments performed in duplicate. Note that, in co-expressing cells Che-1 but not its deletion mutant Che-1-C abrogates the ability of NRAGE to induce cell death. (C) Whole-cell extracts from transfected P19 cells were subjected to western blot analysis. Immunoblotting with anti-Myc monoclonal antibody was performed to visualise expression of Myc-tagged proteins, NRAGE (upper band), Che-1 and Che-1-C (middle and lower bands, respectively). The same samples were monitored for endogenous level of activated caspase-3 using antibody against cleaved caspase-3. Samples were normalised for protein content by reprobing blots with a monoclonal antibody to -tubulin. (D) Caspase-3 activity was measured in P19 cells in which Myc-NRAGE was co-transfected with increasing amounts of Myc-Che-1, as indicated. Caspase activity is shown as the n-fold increase in caspase activation by comparing the levels of luciferase activity of transfected cells with those of untreated control cells. Equal numbers of cells were analysed. Each point represents the mean of three different experiments performed in triplicate.