Over-expression of CLU confers TX-resistance to OVK18 cells. A.Western blotting analysis showing the expression level of s-CLU and mature secreted (40 kDa) CLU in the media in two recombinant OVK18 survivor clones F-1 and F-2 compared with two mock clones M-1 and M-2. The pIRES-hyg-full-length-CLU cDNA expression vector was used for transfection experiments (see Materials and Methods). S-CLU was only detectable in the media of F-1 and F-2 clones. B. Comparison of relative viability of clones F1 and F2 with regard to mock clones M1 and M2 in the presence of different doses of TX. F-1 and F-2 clones show significantly increased viability. Each data point represents the mean of three experiments; bars denote SD; * indicates difference from mock at P < 0.001. C. Quantification of the relative proportions of apoptotic cells by FACS analysis of M-1 and -2 and F-1 and -2 clones in a time-course experiment. Cells were counted, divided into groups in triplicates and challenged by TX at 100 nm for the indicated time periods. Cells were then acquired by FACS calibrator and the apoptotic sub-diploid peak was analyzed and quantified using the Cell-quest software. Significant inhibition of TX-induced apoptosis was observed in the clones stably expressing CLU (F-1 and F-2).