Figure 4

Determination of knockdown efficiency and specificity of siRNAs in the HCC1954 cell system. A. qRT-PCR results showing the knockdown efficiency of 20 nM pools of four siRNAs for each gene in the network (50 nM siRNA for ESR1). ACTB and HPRT1 were used as house-keeping controls. MOCK stands for the samples which were treated only with Lipofectamine transfection reagent. B. Western blots for the determination of knockdowns for the network elements at protein level. Since dimers of ERBB family members are accepted as functional units, we used combinatorial RNAi (knockdown of two genes simultaneously) to produce knockdowns of such dimers. C. qRT-PCR results showing the knockdown efficiency and effect of one member of ERBB receptor family siRNA on the other two members of the family. Both pools of four individual siRNAs per gene and only one individual siRNA per gene were used. The concentration of siRNAs was 20 nM. ACTB was used as house-keeping control. D. Western blots showing the knockdown efficiency and cross reactivity of siRNAs at protein level. β-actin was taken as loading control.