Radiolabeled Isoprenoid Biosynthesis in Plasmodium falciparum

AlbuMAX I Lipid-Rich BSA and RPMI-1640 were purchased from Thermo Fisher Scientific (Leicestershire, UK). Dolichol and dolichyl-P 13–21 were purchased from Avanti (Alabama, USA). [1-(n)-³H] GGOH (14 Ci/mmol; 1 mCi/mL), [1-(n)-³H] FOH (14 Ci/mmol; 1mCi/mL) and L-[4,5-¹⁴C (U)] isoleucine (200–300 mCi/mmol; 0.1 mCi/mL) were purchased from American Radiolabeled Chemicals (St. Louis, USA). SYBR Green I nucleic acid gel stain and SYTO 11 were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, EUA). Sterile stock solutions were prepared at 10 mM for fosmidomycin sodium salt hydrate in water, 2 mM clindamycin hydrochloride in water, 125 mM of GGOH in ethanol and 200 mM of each other non-radiolabelled prenols in ethanol. All other reagents were purchased from Sigma (St. Louis, Missouri USA) or specific companies, as cited in the text. Polyprenyl phosphates were obtained by mild acid treatment of the respective commercial pure pyrophosphates (Sigma; Sigma codes F6892 and G6025) [53 (link)]. For this work, a Cre-LoxP P. falciparum NF54 strain [54 (link)], a generous gift of Moritz Treeck (The Francis Crick Institute, London, United Kingdom), was employed. Erythrocytes and CPDA-1 treated hospital-grade plasma bags for transfusions (AB+) were obtained from the Blood Center of Sírio Libanês Hospital (São Paulo, Brazil).