The role of GSK-3β in the redistribution of p53 induced by sorafenib in the setting of HDM2 blockade. (A). Nuclear and mitochondrial p53 levels in A375 and SKMEL5 cells treated with 10 μM sorafenib (S) and/or 20 μM MI-319 (M). In these studies, c-myc and COX4 were used as markers for the nuclear and mitochondrial subcellular fractions, respectively. (B). (Upper Panel). Nuclear and mitochondrial p53 and AIF levels in A375 cells expressing a tetracycline-regulable GSK-3β shRNA treated with 10 μM sorafenib (S) and/or 20 μM MI-319 (M) with or without doxycycline pretreatment. The downmodulation of GSK-3β in the doxycycline-treated cells was validated by western blot. (Lower Panel). Nuclear and mitochondrial p53 and AIF levels in SKMEL5 and SKMEL5-GSK-3β S9A cells treated with 10 μM sorafenib and/or 20 μM MI-319. (C). Effect of GSK-3β on the pro-apoptotic effects of single agent sorafenib and the sorafenib/MI-319 combination as determined by flow cytometry. Cell death data are presented as the percentage of total cells staining positive for propidium iodide.