Pravastatin

α,β-dehydrolovastatin (DHLV) and its derivatives, lovastatin and α,β-dehydrodihydromonacolin K, were produced by the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178, which was deposited as BCC56851 at BIOTEC Culture collection, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand [23 (link)]. Mevastatin, pravastatin, and simvastatin (Cat# M2537, P4498, and S6196) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12; Cat#12400–024), fetal bovine serum (FBS; Cat#10270–106), trypsin-EDTA (Cat#25300–062), penicillin/streptomycin (Cat#15140–122) were purchased from Thermo Fisher Scientifc Inc. (Waltham, MA, USA). Cholera toxin (CT; Cat#10654, Lot#10067A1) was obtained from List Biological Laboratories, Inc. (Campbell, CA, USA). Heat-stable toxin (STa; Product no.4044297, Lot#1000007536) was purchased from Bachem (Torrance, CA, USA). Dorsomophin or Compound C (AMPK inhibitor; Cat#P5499), CPT-cAMP (Cat#C3912), genistein (Cat#C6649), ATP (Cat#A5394), forskolin (Cat#F6886), Na₃VO₄ (Cat#S6508), CFTR_inh-172 (Cat#C2992), IBMX (phosphodiesterase (PDE) inhibitor; Cat#I5879), Ouabain (Cat#O3125) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MK571 (Cat# 70720) was purchased from Cayman Chemical (Michigan, USA). Other chemicals were purchased from Merck Millipore (Burlington, MA, USA).

With regard to the antioxidative drugs, N-acetyl-L-cysteine (NAC) and amifostine (2-(3-Aminopropyl)aminoethyl phosphorothioate, WR2721, Ethyol) were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France; #9165 and #A5922, respectively). Cells were incubated with these two last drugs separately for 24 h at 37 °C at 10–300 mM NAC or at 24–96 mM for amifostine. The choice of these drug concentrations was motivated by preliminary experiments based on the assessment of γH2AX foci when drug was applied alone to radioresistant fibroblasts. All the conditions leading to the production of more than 2 γH2AX foci per cell were excluded. With regard to the pro-episkevic drugs, cells were incubated with either 1 µM pravastatin (#P4498, Sigma-Aldrich) for 24 h at 37 °C alone or 1 µM zoledronate (#SML0223, Sigma-Aldrich) for 12 h at 37 °C alone (to permit intercomparisons) or together (ZOPRA treatment) [46 (link)]. The choice of these drug concentrations was notably motivated by our previous data published in [46 (link)].

Recombinant pneumolysin (PLY) was kindly provided by Dr. M. Plečkaitytė (Vilnius University, Vilnius, Lithuania) and was generated as previously described [34 (link)]. The statins simvastatin and pravastatin as well as methyl-beta cyclodextrin and melittin were purchased from Sigma Aldrich.

The combination (ZOPRA) of an anti-osteoporosis bisphosphonate (zoledronate) and an anti-cholesterolemic statin (pravastatin) has been shown to accelerate the RIANS [39 (link)]. The ZOPRA treatment was applied under conditions that are described elsewhere. Briefly, cells were incubated with 1 µM pravastatin (#P4498, Sigma-Aldrich France, Saint-Quentin-Fallavier, France) in phosphate-buffered saline (PBS) (#14040-091, Sigma-Aldrich) for 24 h at 37 °C. Thereafter, 1 µM zoledronate (#SML0223, Sigma-Aldrich) in PBS was added to the culture medium, and cells were incubated for 12 h at 37 °C [39 (link)].