Figure 6

Targeting CLU by OGX-011 sensitizes ovarian cancer cells to TX treatment. A.Comparative viability of chemoresistant ovarian cancer cells before and after CLU knock down by OGX-011. 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. KF-TX cells showed enhanced TX-induced toxicity in CLU KD cells versus controls. B. A representative time-dependent DNA histogram (FACS analysis) demonstrating that CLU KD by OGX-011 at 1200 nM enhanced TX toxicity in KF-TX cells. KF-TX cells were transfected either with OGX-011 or control Oligonucleotide and then challenged with TX dose of 200 nM at indicated time periods (24 h, 36 h,48 h and 60 h). B. Results of Annexin V staining of cells pre-treated with OGX-011 at different concecntrations (400, 800 and 1200 nM) then treated with TX (200 nM) for indicated time periods (24, 48 and 72 h). Quantification of the relative ratio of apoptotic cells at different time points indicated the significant enhancement of TX toxicity by OGX-011. The maximum enhancement was obtained by 800 nM OGX-011 while the conc. of 1200 nM did not show further significant improvement in toxicity. D. CLU knock down modulates cellular growth rate of ovarian cancer cells. (1) KF-TX cells showed enhanced growth rate when transfected with CLU siRNA with regard to controls. KF-TX cells either pre-transfected twice with CLU siRNA or control siRNA and then divided in 24 well plat in triplicates. Cell number was counted manually each 12 h (2). Representative clonogenic assay shows that targeting CLU by siRNA (sh-CLU) increased TX-induced clonogenic toxicity in KF cells. In this case, KF cells were either transfected with CLU short hairpin expressing vector (CLU-shRNA) or mock control alone and then cells were challenged by increasing doses of TX starting from 2-5 nM for three weeks. The resistant colonies surviving drug stress were stained by Giemsa after methanol fixation and pictures were taken with a digital camera..