Fig. 4. Overexpression of Gas5 suppresses both the association of GR with cIAP2 GREs and GR-induced cIAP2 mRNA expression.
HeLa cells were transfected with increasing amounts of Gas5-expressing plasmid and treated with 10−6 M of dexamethasone. The ChIP assay and total RNA purification were performed using fractions of these cells. (A) Gas5 suppresses the association of GR with cIAP2 GREs. ChIP assays were performed with the GR antibody against GR LBD (anti-GR Ab) and the cIAP2 promoter fragment that contains tandem GREs was amplified by PCR with an adjusted PCR cycle using a specific primer pair. The bottom blots are Western blots showing GR protein abundance.
(B and C) Gas5 suppresses dexamethasone-induced cIAP2 mRNA expression. Total RNA was purified, and the abundance of the cIAP2 and control RPLP0 mRNAs and of the Gas5 RNA were measured with SYBR Green real-time PCR. Bars represent the mean +/− SEM values of fold induction of the cIAP2 mRNA (B) and the Gas5 RNA (C) expression normalized for the RPLP0 mRNA abundance compared to the baseline (in the absence of Gas5 transfection and dexamethasone). *, p < 0.01, compared to the condition in the absence of Gas5 transfection and in the presence of dexamethasone (n=3).