3114 Nucleic Acids Research, 2009, Vol. 37, No. 9 Figure 1. CK1d silencing decreases the transcriptional activity of ERa and downregulates E2-induced expression of ERa target genes. (A) MELN cells (5 104) were plated in 24-well plates in phenol red-free DMEM containing 10% stripped DSS. Cells were transfected with 5 nM scrambled siRNA (CT siRNA) or with 5 nM CK1d siRNA for 48 h and incubated with or without E2 (10 nM) for 24 h. ERE-dependent gene expression was quantiﬁed by measuring luciferase activity, given as fold of control. Error bars represent SD of two experiments. (B) Quantitative real-time RT–PCR validation of down-regulation of CK1d mRNA levels after treatment with 5 nM siRNA. MCF7 cells (2 105) were plated in 6-well plates in phenol red-free DMEM containing 10% DSS. Cells were transfected either with 5 nM CT siRNA or with 5 nM siRNA targeting CK1d and treated or not (vehicle) with E2 (10 nM) for 24 h. Cells were harvested and total RNA was extracted and used to synthesize cDNA by reverse transcription, as