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 or control siRNA twice. Twenty-four hours after last transfection, cells were treated with TX. Seventy-two hours after drug addition at 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. 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).