Fig 6.

Hypoxia or CoCl₂ treatment enhances the RTA-mediated inducible LANA promoter activity (A) Schematic representation of the RTA-inducible LANA promoter region showing the presence of RTA response elements (RRE) in relation to the putative HRE sequences. RRE are shown as rectangular boxes filled with black color. (B) Hypoxia or CoCl₂ enhance RTA-inducible LANA promoter activity. Hep3B cells were transfected with 400 ng empty vector or an RTA expression plasmid along with a fixed amount (300 ng) of pGL3-LANA(i)p-luc. At 24 h posttransfection, cells were exposed to hypoxia or treated with CoCl₂ (100 μM) for 16 h. At the end of incubation period, cells were lysed and promoter activity was determined as described in the legend to Fig. 1. All results are normalized to the empty vector control in normoxia. CoCl₂ or hypoxia alone induced the inducible LANA promoter by approximately 1.6-fold and 2-fold, respectively. All values represent means of triplicate determinations of a representative experiment out of two. Error bars represent the standard deviation. (C) HIFs cooperate with RTA to increase the LTi LANA promoter activity. Hep3B cells were cotransfected with 300 ng of pGL3-LANA(i)p-luc along with 200 ng of degradation-resistant HIF-1α or HIF-2α and/or 200 ng RTA expression plasmid as shown. The total amount of DNA was normalized where appropriate using the pcDNA control (200 or 400 ng). At 48 h posttransfection in normoxia, cells were lysed and promoter assay was assessed as described in the legend to Fig. 1. Fold activation was calculated by normalization to the pcDNA control. All values represent means of triplicate determinations. Error bars represent the standard deviations.