Figure 8.

CSTF2 overexpression promotes the 3′-UTR shortening of the HuR transcript in TAMR breast cancer cells. A, CSTF2 protein levels in MCF7 control and TAMR cells were determined using Western blotting, with β-actin as an input control. B, the levels of HuR mRNA isoforms terminated at different PASs in MCF7/TAMR transfected with either scrambled siRNA (NC) or siCSTF2 were assessed by RT-qPCR analysis. C, cDNA was synthesized from total RNA isolated from MCF7/TAMR/siNC or MCF7/TAMR/siCSTF2 cells using anchor oligo(dT) from the 3′-RACE kit (Roche Applied Science). The resulting amplification products were analyzed using agarose gel electrophoresis. D, the CSTF2 and HuR protein levels in MCF7 TAMR cells transfected with either siNC or siCSTF2 were determined using Western blotting with β-actin as an input control. E and F, MCF7/TAMR cells transfected with either siNC or siCSTF2 were seeded in 6- or 96-well plates and treated with 1 mm 4OH-tamoxifen or ethanol vehicle. E, at the indicated time points, cells were trypsinized, and total cells were counted. F, at 96 h after plating, an MTT assay was performed to determine the viabilities of the MCF7/TAMR/siNC and MCF7/TAMR/siCSTF2 cells. *, p < 0.05. Error bars, S.E.