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. Author manuscript; available in PMC: 2013 Dec 30.
Published in final edited form as: Mol Cancer Ther. 2010 May 25;9(6):10.1158/1535-7163.MCT-09-0880. doi: 10.1158/1535-7163.MCT-09-0880

Figure 3.

Figure 3

Expression of DN-AKT disrupts sCLU expression. A, DU145-DR tumor cells were transfected with DN-AKT and cultured for 48 h in docetaxel-containing medium. Control transfection was with the empty vector PCDNA3. Transfection efficiency was monitored by Western blot analysis for total AKT. pAKT assessment indicated that the overexpressed DN-AKT is not phosphorylated, as expected, due to mutation at the critical threonine and serine sites, thus showing equal levels of pAKT in control-transfected and DN-AKT–transfected cells. Downstream AKT function was assessed by analysis of GSK-3β phosphorylation, which is a known AKT target. DN-AKT effectively suppressed GSK-3β phosphorylation and correspondingly suppressed sCLU expression. B, the above experiment was repeated on PC3-DR cells and produced confirmation of the loss of AKT to result in loss of pGSK-3β and sCLU. C, analysis of sCLU gene expression was done by RT-PCR on DU145DR and PC3-DR cells that were either treated with DMSO or API-2, or transfected with control PCDNA3 vector or DN-AKT. Both API-2 treatment and DN-AKT transfection markedly suppressed CLU mRNA expression in DU145-DR and PC3-DR tumor cells compared with their respective controls. Equal loading was monitored by GAPDH mRNA levels.