Fig. 3.

FOXO3a stimulates transcription from iP2 in the context of the MIE genomic locus. (A) A plasmid containing the regulatory and coding regions for the HCMV IE1 and IE2 genes (pSVH) or a variant of pSVH lacking the core MIEP (pSVHΔMIEP) was transfected into HeLa cells together with a FOXO1 or FOXO3a expression plasmid, or an empty expression vector (EV) control. Cells were harvested at 24 h after infection, and cell lysates were analyzed by Western blot using the indicated antibodies. Results are representative of three independent experiments. (B) Each FOXO consensus site in iP2 was mutated in the context of the pSVHΔMIEP plasmid, either alone (mut1, mut2, and mut3) or in combination (mut123). Cells were transfected with the indicated plasmid along with a FOXO3a expression plasmid (FOXO3a) or the EV control and harvested 24 h later. Cell lysates were analyzed by Western blot using the indicated antibodies. The results are representative of three independent experiments. (C) Cells were transfected and harvested as in B. RNA was extracted and analyzed by qRT-PCR using primers specific for UL123 (encoding IE1), UL122 (encoding IE2), iP1, or iP2. The graphs show the fold change in RNA abundance compared to cells transfected with pSVHDMIEP and the empty expression vector control (n = 3; *P < 0.05; NS = not significant).