Silica gel 60a

Compound RA1 was purified by column chromatography packed with silica gel 60A (60–200 µm, Acros) and the purification process monitored by thin layer chromatography (TLC) on aluminium plates coated with 60F₂₅₄ (0.2 mm, Merck). Spots were visualized with UV light at 254 nm or ninhydrin (Sigma-Aldrich). The eluent system used was hexane/ethyl acetate (6:4, v/v). Analytical RP-HPLC was performed on a Shimazo Prominence-I LC-2030 C 3D and elution of the compounds was monitored by absorbance at 254 nm. Compound purity was assessed using a XBridge C18 3.5 µm 4.6 × 250 mm column (Waters) and a linear gradient of 10–60% acetonitrile (containing 0.08% TFA) in water (containing 0.08% TFA) for compounds RA2 and RA3 over 40 min at a flow rate of 1 mL/min.

To measure PLD activity, PHK infected with different recombinant retrovirus were plated in 100 mm culture dishes and allowed to reach about 80% confluence. Cells were then pre-labeled with [³H] myristic acid (Perkin-Elmer, Waltham, MA, USA) (6 μCi, 30 Ci/mmol) in 4 ml of culture medium for 5 h. PLD activity was determined by measuring the formation of [³H] phosphatidylbutanol (PtBt), the product of PLD-mediated trans-phosphatidylation, in the presence of 0.8% 1-Butanol (Sigma-Aldrich, St. Louis, MO, USA). Lipid extraction and characterization were performed by thin layer chromatography (TLC). Briefly, lipid extracts were counted in a scintillation counter to normalize all samples. Approximately 500.000 CPM of each sample were spotted onto TLC plates (silica gel 60A, Fisher Scientific, Fair Lawn, NJ, USA). TLC were developed with the upper organic phase of a mixture containing ethyl acetate:iso-octane:glacial acetic acid:H2O (1.1:0.5:0.2:1, vol/vol) and allowed to dry before being sprayed with En3hancer Spray (Perkin Elmer, IL, USA). Phosphatidilbutanol was detected by autoradiography with Kodak BioMax MR Film (Kodak, Rochester, NY, USA).
PLD activity in control and HPV gene-expressing primary human keratinocytes after pRb downregulation was determined using the Phospholipase D Assay Kit (#700590, Cayman Chemicals, Ann Arbor, MI, USA) according to the manufacturer’s instructions.