Figure 7. Uridine and orotate supplementation reverse the effects of redoxal on A3G protein levels in the absence of Vif.

A. Uridine supplementation diminishes redoxal-induced augmentation of cellular A3G protein levels in the absence of HIV-1 Vif. 293T cells were co-transfected with 100 ng A3G-3xHA and 1000 ng pCDNA3.1 (empty vector) plasmids. At 5 h post transfection, the media was replaced with fresh media supplemented with DMSO or 1250 nM redoxal and increasing doses of uridine (0, 10, or 50 μM). At 40 h post transfection, cells were lysed. A3G and β-tubulin protein levels in cell lysates were analyzed by Western blotting. Results are representative of two independent experiments. B. Orotate supplementation diminishes redoxal-induced augmentation of cellular A3G protein levels in the absence of HIV-1 Vif. 293T cells were co-transfected with 100 ng A3G-3xHA and 1000 ng pCDNA3.1 (empty vector) plasmids. At 5 h post transfection, the media was replaced with fresh media supplemented with DMSO or 1250 nM redoxal and supplemented with increasing doses of orotate (0, 0.5, or 2.0 mM). At 40 h post transfection, cells were lysed. A3G and β-tubulin protein levels in cell lysates were analyzed by Western blotting. A3G blots are shown for two exposure times (20 and 60 sec). Results are representative of two independent experiments. Values shown below blots in A and B represent relative A3G protein levels in treated cells determined by densitometry using ImageJ software and normalization to A3G protein levels in untreated cells (DMSO control). C. Pyrimidine depletion by redoxal decreases cell viability. Viability of treated cells described in Fig. 8B was measured using CellTiter-Glo^® Luminescent cell viability assay. Results are representative of two independent experiments. Shown are means ± SD of duplicate samples from independent wells (p-values from student’s t-test).