Figure 6.

Effect of knocking down PTEN on icIL-1RA and sIL-1RA mRNA stability and gene transcription in OFs. Eighty percent of confluent cultured cells were transfected with specific siRNAs targeting PTEN or scrambled oligonucleotide control. After 48 hours, monolayers were untreated or treated with bTSH (5 mIU/mL) for 12 hours. A, Stability of icIL-1RA and sIL-1RA mRNA was determined and analyzed by real-time RT-PCR as described under Materials and Methods. All cultures were pretreated with bTSH for 12 hours, and then at time = 0 DRB (50 μg/mL) was added to the cultures. Half the wells were then treated with bTSH for the times indicated along the abscissa while the others did not receive TSH (control). B, ChIP assays were performed using formaldehyde cross-linked samples from untreated cultures and those treated with bTSH. Promoter DNA for either icIL-1RA or sIL-1RA was generated by RT-PCR, and RNA Pol II occupancy was normalized to GAPDH and was expressed as mean ± SD of fold-change of three independent experiments. Data are expressed as mean ± SD of three replicates.