Figure 6.

siRNA-mediated knockdown of CNOT1 results in increased induction of ERα target genes TTF1 and c-Myc. (A) Knockdown of CNOT1 by siRNA. Two different double-stranded RNA oligonucleotides were used. A nontargeting control was also included. Protein lysates were prepared 72 h after transfection and analysed by immunoblotting using the indicated antibodies. (B) Knockdown of CNOT1 does not affect protein levels of known ligand-dependent cofactors of ERα. (C) Increased TFF1 (pS2) mRNA induction in cells treated with siRNAs directed against CNOT1. mRNA levels were determined in MCF-7 breast cancer cells before and after treatment with vehicle or E₂ by RT–PCR using GAPDH as a reference. Subsequently, the fold induction was calculated. (D) Increased c-Myc mRNA induction in cells treated with siRNAs directed against CNOT1. Levels of c-Myc mRNA were determined using GAPDH as a reference. (E) E₂-mediated induction of TFF1 mRNA is increased in cells with depleted CNOT3 mRNA. MCF-7 cells were transfected with siRNAs against the Ccr4-Not subunits CNOT2, CNOT3, and CNOT9/RQCD1. (F) Differential effects on c-Myc mRNA induction caused by depletion of the Ccr4-Not subunits CNOT3 and CNOT9/RQCD1. Errors of the mean are indicated in panels (C)–(F).