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. 2009 Mar 4;83(10):4871–4883. doi: 10.1128/JVI.02286-08

FIG. 5.

FIG. 5.

Necdin downregulates the HRE sequence containing promoter activity, whereas EBNA3C and Nm23-H1 rescue it from Necdin-mediated downregulation. In all experiments, 10 million 293T cells were transfected with 5.0 μg of pGL3-HRE reporter construct (except in panel b, where 5.0 μg of pGL3-MHRE was used) along with other expression constructs as explained below. All the results are expressed as means ± standard errors of the means from three separate experiments performed in duplicate or triplicate. (a) Five micrograms of pA3M-Nm23-H1, pA3M-EBNA3C, pA3HA-Necdin, or a vector control was transfected. The results show that Necdin downregulates HRE promoter activity (lane 3), whereas EBNA3C and Nm23-H1 (lanes 2 and 4) do not have any significant direct effect on the activity of HRE promoter. (b) Five micrograms of Nm23-H1, EBNA3C, Necdin, or a vector control was transfected. The results show that Necdin did not induce any effect on MHRE promoter activity (lane 3), suggesting that the effect of Necdin on HRE is specific. (c) Cells were transfected with 0, 2.5, 5.0, 7.5, or 10.0 μg of pA3HA-Necdin. The results show that the downregulation of HRE by Necdin is dose dependent (lanes 2 to 5). (d) Five micrograms of pA3HA-Necdin, along with 0, 0.1, 0.2, 0.3, or 0.5 μg of pA3M-Nm23-H1, was transfected. The results show that Nm23-H1 was able to rescue the HRE-dependent transcription from Necdin-mediated downregulation in a dose-dependent manner (lanes 3 to 6). (e) Five micrograms of pA3HA-Necdin, plus 0, 0.1, 0.2, 0.3, or 0.5 μg of pA3M-EBNA3C, was transfected. The results show that EBNA3C was able to rescue the HRE-dependent transcription from Necdin-mediated downregulation in a dose-dependent manner (lanes 2 to 6). (f) Five micrograms of pA3HA-Necdin with or without 5.0 μg EBNA3C was transfected. The results confirmed that EBNA3C is able to rescue activity of the VEGF promoter from Necdin-mediated downregulation (lane 3).