Phosphoimager screen

The cross-linked miniRTIC, RT, and vRNA–tRNA used in ³²P activity assays were prepared as described above. miniRTIC (50 nM) was preincubated in 50 mM Tris-HCl, pH 8.0, 50 mM KCl, and 6 mM MgCl₂ in a 37 °C water bath for 5 min. For drug conditions, nevirapine (50 nM) and efavirenz (50 nM) were separately incubated with miniRTIC (50 nM) under the same conditions. Free vRNA–tRNA (50 nM) and RT (250 nM) was also preincubated under the same conditions. Incorporation reactions were initiated by adding a mixture of α-³²P-dCTP (40 nM) and dCTP (50 μM). Reactions were quenched at various time points between 5 s and 4 h with the addition of EDTA and SDS loading buffer. The reaction sets for each condition (free, x-link, x-link + nevirapine, x-link + efavirenz) were run on a 12% SDS-PAGE gel, dried, and exposed for 18 h on a phosphoimager screen (Molecular Dynamics) and each gel was individually scanned with a Storm 860 (Molecular Dynamics)^{12 (link),13 (link)}. Bands were quantified using ImageQuant. The intensity was normalized to the highest intensity for the individual time-course assays after background subtraction (set to 1). The miniRTIC and free vRNA–tRNA with RT were repeated five times. Nevirapine and efavirenz conditions were each repeated three times. Plotting and curve fitting was performed using GraphPad Prism8.

E. coli strain BL21(DE3) transformed with the expression plasmid pET28::CT776 (AasC) was grown in LB rich medium at 37 °C to an OD₆₀₀ of about 0.4 and expression was induced for 1 hour with 0.5 mM IPTG. Un-induced cells were grown to an OD₆₀₀ of about 0.7. Cells were harvested and were lysed with a FrenchPress in 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM DTT and 10% glycerol. Reactions were performed in 450 µl of 50 mM Tris-HCl pH 7.4, 20 mM MgCl₂, 3 mM DTT, 10 mM ATP and 5 µM [¹⁴C]C₁₆-OH, which was suspended in 20 µl 0.55 mM Triton-X100. When mentioned, CoASH was added at 0.5 mM and 1-acyl-GPC at 20 µM. Reactions were initiated by the addition of 50 µg of protein extract and incubated at 30 °C. At the indicated times, 100 µl of the reaction was removed and transferred into 375 µl of CHCl₃/MeOH (1/2; v/v) and lipids were extracted with the Bligh and Dyer method. Stock solution of triacsin C and rosiglitazone G were made in 98% ethanol at a 100x concentration and 4.5 µl was added before initiating the reaction. Control reactions were performed in the presence of 4.5 µl of 98% ethanol. Lipids were separated on TLC plates as described above. TLC plates were air-dried and exposed to a PhosphoImager screen (Storm 840, Molecular Dynamics). Quantification of the rate of formation of [¹⁴C]PLs was performed with ImageQuant software after subtraction of the plate background.

Each reaction (total volume, 10 μl) contained 1 μg recombinant protein, 0.25 mM cold ATP, 5 μCi γ³²P-ATP and 350 ng CDK1–cyclin B recombinant human protein (Thermo Fisher PV3292) in kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% Brij-35) supplemented with PhosSTOP (Sigma-Aldrich) and Complete EDTA-free proteases inhibitors (Sigma-Aldrich). The samples were incubated at 30°C for 10 min. The reactions were stopped by adding 3× Laemmli sample buffer and boiling at 95°C for 5 min. The samples were then resolved on a 10% acrylamide gel. The gel was stained with Coomassie Blue, dried and exposed to a phosphoimager screen (GE Healthcare). The results were analysed with a Typhoon FLA 9500 (GE Healthcare).

1 μg of isolated mtDNA was linearized with SacI, precipitated and dissolved in 10 mM Tris HCl pH 7.5. The DNA was hydrolyzed with 0.3 M NaOH at 55°C or digested with RNase H2 (New England Biolabs) at 37°C for 2 h. Samples were run on a 0.8% agarose alkaline gel (30 mM NaOH, 1 mM EDTA) at 25 V, 4°C for 20 h and blotted onto Hybond-N+ membrane (Amersham, GE Healthcare). Single-stranded probes were end-labelled with γ-³²P ATP using T4 polynucleotide kinase (Thermo Scientific) following the manufacturer’s protocol. Double-stranded probes were generated by labelling an approximately 500 bp PCR product with α-³²P dCTP using Prime-It II Random Primer Labeling kit (Agilent Technologies). Hybridization was for 16 h at 42°C for ssDNA probes and at 65°C for dsDNA probes. The membrane was exposed to a PhosphoImager screen and scanned in a Typhoon laser scanner (GE Healthcare). The radioactive intensity was quantified using ImageJ software and plotted on a distribution plot. The median size of alkali-treated products was determined from the distribution of the radioactivity intensity and related to the size marker that was run in parallel [7 (link)].