TX resistant ovarian cancer cells, KF-TX, were transfected either with siRNA or OGX-011. CLU gene mRNA was amenable to siRNA transfection at doses of 100 and 200 nM (Figure 5A.1) and to OGX-011 at doses of 400, 800, 1000 and 1200 nM as well (Figure 5A.2). We then considered 200 nM of siRNA and SCH772984 solubility dmso control siRNA and 1200 nM of OGX-011 and control oligodeoxynucleotide to be used in further experiments because they maximally reduced CLU expression. Figure 5 Targeting CLU by siRNA or OGX-011 sensitizes ovarian cancer cells to TX treatment. A. Western blotting showing the

efficacy of siRNA transfection or OGX-011 in s-CLU depletion in KF-TX cells. (1) CLU expression after two sequential transfections with siRNA against CLU (see materials and methods) at 100 and 200 nM are compared with control siRNA at 200 nM. Transfection at 200 nM knocked down about 90% of target CLU (far right panel). The basic expression level without any transfection had not

been affected neither by transfection reagents (data not shown) nor by control siRNA transfection (far left panel). (2) CLU expression after two sequential transfections with OGX-011 (see materials and methods) at 200-1200 nM are compared with control oligonucleotides. OGX-011 transfection at 800, 1000 and 1200 nM significantly knocked down CLU expression (far right panels). B. Comparative viability of different ovarian cancer cells before and after CLU knock down are. Cells were cultured in 96-well plates, then transfected either with CLU-siRNA selleck screening library or control siRNA twice. Twenty-four hours after last transfection, cells were treated with TX. Seventy-two hours after drug addition at selleck compound indicated doses, cell viability was estimated. Both KF-TX cells (1)

and SKOV-3-TX (2) showed enhanced TX-induced toxicity in CLU KD cells versus controls. MycoClean Mycoplasma Removal Kit C. Time-dependent FACS analysis demonstrating that CLU-siRNA enhanced TX toxicity in KF-TX cells. KF-TX cells were transfected either with CLU-siRNA or control siRNA and challenged with TX dose of 200 nM at indicated time periods. Representative DNA histograms show the apoptotic response to TX with and without CLU-siRNA transfection (1). Annexin V staining of cells treated as in panel (1). Time-course quantification of the relative ratio of apoptotic cells at different time points in the presence of CLU siRNA or controls when cells were challenged with TX (2). To evaluate the benefits of targeting s-CLU in sensitizing ovarian cancer cells to TX, cellular viability of KF-TX under a dose dependent fashion of TX treatment was studied in both CLU-siRNA and control-siRNA (cont-siRNA) transfected cells. Under these experimental conditions Figure 5B.1 shows significant reduction in cell viability of KF-TX, pre-treated with CLU-siRNA, under different doses of TX than those pre-treated with control-siRNA then TX.