B. Western analysis showing s-CLU expression in cell extracts (upper panel) and culture media (lower panel) after 48 h treatment with TX. CLU increased in TX-sensitive KF cells at different doses while CLU secretion was inhibited. At difference, expression and secretion of CLU was unchanged in the TX-resistant cells. Only at very high concentrations of TX a consistent down-regulation of s-CLU in the media was detectable. Ponceau S staining of

the blot is provided to show equal loading of the protein samples because Actin and tubulin are responding to TX. The data shown are representative of four independent experiments. Overexpression of s-CLU confers resistance to learn more TX in vitro To confirm the cytoprotective role of s-CLU in vitro, we established two cell clones stably expressing full-length

CLU (a gene able to express s-CLU) from the OVK18 cells with low endogenous CLU, OVK18-s-CLU-1 (F-1) and OVK18-s-CLU-2 (F-2). As shown in Figure 4A, very limited endogenous CLU is expressed and secreted by parental OVK18 cells, while CLU is detectable in both F-1 and F-2 clones as precursor and secreted form in cell extract and media. When cell viability of both clones was assayed under progressively increasing TX doses, it was significantly higher than mock controls (M-1 and M-2 (p < 0.05; Figure 4B)). Figure 4C summarizes the result of FACS analysis of F-1/F-2 clones compared to M-1/M-2. F-1 and F-2 showed a significantly lower cell death as assessed as sub-diploid peak, under TX stress when compared to M-1 and M-2. These data confirmed the cytoprotective effect of s-CLU Vadimezan supplier in ovarian cancer cells. Figure 4 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 Niclosamide 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 Nutlin 3a 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).