Stavudine

Cells were plated in a 6-multiwell plate (MW-6; Corning Incorporated, Kennebunk, ME, USA) (8x104 cells per well) and cultured in the appropriate medium for each type, following the same procedure (Fig 1). Cells were maintained for 24 hours in the appropriate culture medium before the medium was supplemented with each tested condition: 3,8-Diamino-5-ethyl-6-phenylphenanthridinium bromide (Ethidium bromide, EtBr) (Sigma-Aldrich), Rhodamine 6g (Rd6g) (Invitrogen, Waltham, MA, USA), 1-methyl-4-phenylpyridinium (MPP⁺), Zidovudine (AZT, 3´-azido-3´-deoxythymidine) or Stavudine (d4t; 2'-3'-didehydro-2'-3'-dideoxythymidine) (Sigma-Aldrich) (Table 1). Culture media were always supplemented with uridine (50 mg/ml). Drug stocks were prepared following the manufacturers' instructions and replenished at each medium change. The stocks were stored at -20°C between doses and new stocks were prepared before each experiment.
In all the experiments, cells with no treatment were used as controls to determine the baseline levels of mtDNA for each cell type. Cells cultured with EtBr were used as positive controls to assess the capacity of the different cell lines to survive with very low levels of mtDNA. 143B.TK-Rho-0 cells were used as a reference for the mtDNA content characteristic of a standard Rho-0 line.

Parental fly crosses were settled on standard cornmeal added of the below-listed drugs with the reported final concentration: stavudine 10 μM, azidotimidine 10 μM, tenofovir 10 μM, abacavir (#SML0089 Sigma) 10 μM, rilpivirine (#10410 Sigma) 10 μM, enoxacin (#AB143281 Abcam) 10 μM, and lamivudine (#L1295 Sigma) 10 μM. For each drug, a vehicle-only control group was arranged. Parental flies have been maintained 24 h in the tubes to allow the embryo laying. Synchronized embryos were grown to obtain third instar larvae to be tested for mobility or to be analyzed for NMJ morphology.