Fig 7. Monitoring ara-CTP and gem-TP concentrations in MDMs.

A) Monocyte-derived macrophages (MDMs) were pretreated with virus-like particles (VLP) one day prior to replacing the medium with fresh medium plus compounds: 10 μM of ara-C (cytaribine-¹³C₃) or 10 μM of gemcitabine. Whole cell lysates were collected at 0, 24 and 48 h post VLP addition. Lysates were analyzed by immunoblotting for SAMHD1 and GAPDH, loading control. SAMHD1 protein levels were reduced at 24 h after Vpx+ VLP exposure. Two human primary MDM donors are shown. Cellular nucleotide extracts were generated at 4, 12 and 24 h post drug addition from treated MDMs. The intracellular concentrations of B) gem-TP (2',2'-diF-dCTP) and (C) ara-CTP were quantified from the extracts using HPLC-MS/MS analysis. Data are plotted as pmol/million cells (y-axis) vs. time (h) (x-axis). Gem-TP is a significantly lower (**, p < 0.01; T test) in the Vpx+ VLP treated MDMs at 4 h after drug addition. However, the rate of gem-TP decay is comparable between the two groups, suggesting that gem-TP degradation is SAMHD1 independent. Ara-CTP concentrations are significantly higher (***, p < 0.001) at 4, 12 and 24 h for the Vpx+ VLP treated MDMs. Moreover, the rate of decay of ara-CTP is slower in Vpx+ VLP treated group, suggesting ara-CTP turnover is SAMHD1 dependent. Data are from two independent donors tested in duplicate.