Tenofovir

Tenofovir (CAS 147127-20-6; T018500, Toronto Research Chemicals, Canada) was dissolved in phosphate-buffered saline, 7% dimethyl sulfoxide, and 5% 5N sodium hydroxide to result in a 767 mM stock solution, and further diluted in culture media for addition to keratinocyte cultures in concentrations ranging from 0.05 to 32 mM. Based on initial titration experiments in which we measured the intracellular concentration of Tenofovir diphosphate, the active cellular metabolite of Tenofovir, by liquid chromatography-tandem mass spectrometry (performed in the laboratory of Dr Craig Hendrix, Johns Hopkins University), concentrations in the culture media at the lower end (0.05–0.5 mM range) were estimated to be equivalent to the active concentrations likely achieved by topical Tenofovir 1% gel (35 mM) in mucosal epithelial cells in vivo (Hendrix et al., 2013 (link); Louissaint et al., 2013 (link)). In all experiments, control keratinocytes were cultured in parallel without Tenofovir. Keratinocytes were harvested at pre-determined time points, split into several aliquots, and pelleted. Pellets were frozen and stored at −80°C either as dry pellets for protein assays or after suspension in RNAprotect Cell Reagent (Qiagen) for RNA assays.

NVR 3-778 was synthesized by WuXi AppTec (Wuhan, China). Lamivudine, tenofovir, tenofovir disoproxil fumarate, and entecavir were purchased from Toronto Research Chemicals (Toronto, Canada).

[¹⁴C]Tetraethylammonium (specific activity = 55 mCi/mmol) and [³H]efavirenz (specific activity = 5 Ci/mmol) were purchased from American Radiolabelled Chemicals (Missouri, USA). Lopinavir was a gift from Abbott (Illinois, USA). Raltegravir sodium salt was a gift from Merck (New Jersey, USA). Etravirine was a gift from Janssen (Buckinghamshire, UK). [¹⁴C]Darunavir (specific activity = 39.19 mCi/mmol), non-radiolabelled darunavir and non-radiolabelled rilpivirine were gifts from Tibotec (Mechelen Belgium). Atazanavir was a gift from Bristol-Myers Squibb (New York, USA). Nevirapine was a gift from Boehringer Ingelheim (Berkshire, UK). Ritonavir was a gift from Abbott (Illinois, USA). Tenofovir, efavirenz and lamivudine were purchased from Toronto Research Chemicals (Toronto, Canada). Amprenavir was a gift from GlaxoSmithKline (Middlesex, UK). Ultima Gold scintillation fluid was purchased from Perkin Elmer (Boston, USA). All other drugs and reagents were obtained from Sigma (Poole, UK).