Amplicon-Dependent CCNE1 Expression Is Critical for Clonogenic Survival after Cisplatin Treatment and Is Correlated with 20q11 Gain in Ovarian Cancer
Figure 2
siRNA mediated knock-down of 19q12 genes in ovarian tumor cell lines.
(A) Experimental schematic for combined siRNA transfection and drug treatment. (B) qPCR heatmap showing log2 gene expression ratio to untransfected OVCAR-3 (top) and SK-OV-3 (bottom) cells for each siRNA (columns) at gene targets (rows) with or without cisplatin treatment 72 hours after transfection. (C) Cell viability normalized to no siRNA control cells after transfection with each siRNA. Statistical significance (t-test) calculated by comparison to non-silencing (NS) siRNA in the same cell line using data from three independent MTS assays performed with triplicate wells per condition. Average normalized absorbance at 490 nm and SEM plotted (n = 3), **p-value <0.01. (D) Proportion of apoptotic cells assessed by TUNEL staining in no siRNA control cells or after transfection with NS or CCNE1 targeted siRNA. Data shown from three independent experiments with duplicate wells analyzed per condition. Average percentage of apoptotic cells and SEM plotted (n = 3). ***p-value <0.0001 (Chi squared) calculated by comparison of total cell counts between OVCAR-3 cells treated with a CCNE1 siRNA and all other conditions. (E) CCNE1 protein expression by western blot to confirm siRNA-mediated CCNE1 knockdown at experimental endpoint. (F) Cell viability in additional cell lines after transfection with CCNE1 or non-silencing siRNAs normalized to each cell line with no siRNA added. Statistical significance (t-test) calculated by comparison to NS siRNA in the same cell line. Average normalized absorbance from MTS assay and SEM plotted (n = 3); *p-value <0.05, **p-value <0.01.