Figure 8. Effect of insulin-based culture supplement or serum on HIOMT promoter activity

Activation of the chicken HIOMT promoter by newborn calf serum (NCS) and by the insulin-transferrin-selenium culture supplement (ITS) was assessed. E7 retinal cells were cultured in medium supplemented with either 10% newborn calf serum (NCS) or 10% inactive fetal calf serum (i.e., FCS that did not induce northern blot-detectable levels of HIOMT mRNA expression) or with 10% inactive FCS plus insulin-transferrin-selenium (ITS). The cells were transfected with plasmids containing the firefly luciferase reporter gene alone (pGL3-Basic) or under control of either 3 kbp or 225 bp of the chicken HIOMT promoter (pGL3-HIOMT1 and pGL3-HIOMT2, respectively). To correct for variations in transfection efficiencies, all experimental groups were cotransfected with a second plasmid (pRL-SV40) containing the renilla luciferase reporter gene under control of the SV40 promoter. The levels of luciferase activities were measured 24 h post-transfection. The ratios of firefly/renilla luciferase activities were calculated and were expressed relative to pGL3-Basic. Histograms represent the mean (3 in each experimental group) from one representative experiment; error bars represent the SEM. Similar results were obtained in four independent experiments.