Figure 3.

SPOP inhibits progesterone-induced transcription. A. 293T Cells were transfected with MMTV luciferase reporter, and Myc-PRA, or Myc-SPOP constructs, alone or in combination as indicated. After 24 hr, cells were treated with vehicle ethanol (EtOH) or progesterone (10 nM) for 24 hr, and the luciferase activities were measured by luminometer; B. T47D cells were transfected with control siRNAs or SPOP siRNAs, After 24 hr, cells were co-transfected with pMMTV luciferase reporter for 24 hr, and then treated with vehicle ethanol (EtOH) or progesterone (10 nM) for 24 hr, and the luciferase activities were measured by luminometer; C. T47D cells were transfected with control siRNAs or SPOP-specific siRNA. After 48 hr, cells were then treated with the vehicle ethanol (EtOH) or progesterone (10 nM) for 18 hr. The mRNA level of PR target genes (KFBP5, Cyclin D1 and HSD2) was measured by qRT-PCR methods. The mRNA level of GAPDH was used for normalization. The mean values (S.D.) of three independent experiments are shown. * indicates statistical significance (*, p < 0.01); D. T47D cells were transfected with control or Myc-SPOP or SPOP-ΔBTB mutant constructs. After 24 hr, cells were treated with vehicle ethanol (EtOH) or progesterone (10 nM) for 24 hr. Cell lysates were prepared for WB analyses; E. T47D cells were transfected with control siRNAs or SPOP-specific siRNAs. After 48 hr, cells were then treated with the vehicle ethanol (EtOH) or progesterone (10 nM) for 24 hr. Cell lysates were prepared for WB analyses.