Figure 2

RITA did not increase the expression of p53 targets in HMCLs. A. Cells were incubated for 24 hours with different concentrations of RITA or nutlin3a i.e., 150 nM and 4 μM in MDN, respectively, 3 μM and 10 μM in NCI-H929, respectively, 600 nM and 10 μM in NAN8, respectively, and, 20 nM and 10 μM in XG5. Cells were harvested and either lysed following protein expression analysis using western blotting as indicated in the figure, or stained with anti-DR5-PE or control-PE mAbs, and the fluorescence was analyzed using a FACSCalibur. One representative experiment of 3 is shown. DR5 expression fold-change (i.e., DR5 ratio of RITA- or nutlin3a-treated cells divided by ratio of control cells) induced by RITA or nutlin3a is indicated within cytograms. B. Cells were incubated for 3 h (or 24 h) with 10 nM (XG5) or 100 nM (MDN, KMS12PE) RITA or nutlin3a (5 μM for MDN and 1 μM for KMS12PE and XG5). The proteins from the nucleus, mitochondria and cytosol were extracted using the Cell Fractionation Kit-Standard (Abcam, Cambridge, UK).