Myristic acid azide

Parasites were washed with PBS three times and 1 x 10⁷ cells were incubated at 37°C in the case of ICA and TCT, and at 28°C in the case of Epi, in 1 mL of 2% delipidated BSA-DMEM. After 30 min, 100 μM Click-IT myristic acid, azide (Life Technologies, Thermo Fisher Scientific) was added from 50 mM stock solutions in dimethyl sulfoxide (DMSO). The same volume of DMSO was used as a negative control. Parasites were further incubated for 6 h. For confocal microscopy analysis, they were processed as described above. After permeabilization (0.1% Triton X-100 in PBS for 10 min, at room temperature), cells were washed three times with 3% BSA in PBS. “Click” reaction between the myristic acid azide and Alexa Fluor 488 alkyne (Invitrogen) was performed according to the manufacturer instructions using the Click-iT Cell Reaction Buffer Kit (Invitrogen). Parasite DNA was labeled with DAPI. Samples were visualized and images acquired as described above.

Restriction endonucleases were obtained from MBI-Fermentas, Pfu DNA polymerase was from Stratagene and DNA ligases were from New England Biolabs (NEB). E. coli strain NEB 5-α was used for cloning of cDNAs and amplification of plasmids. Oligonucleotides used in the generation of expression constructs, MitoTracker Green FM, myristic acid azide (12-azidododecanoic acid; AzMyr), Alexa Fluor 488 DIBO Alkyne, cell culture reagents, and Lipofectamine LTX (with PLUS™ reagent) were obtained from Invitrogen. Reagents for SDS-PAGE and western blotting were from Bio-Rad. The peptide substrate corresponding to the N-terminal of human HK1S (²GQICORES⁹-CONH₂) was custom synthesized and obtained from the Institute for Biomolecular Design (University of Alberta). Myristoyl-CoA (lithium salt) was purchased from Sigma-Aldrich. PCMV6-Entry vector, anti-DDK monoclonal antibodies and anti-DDK Agarose beads were purchased from Origene. Anti-mouse antibodies linked to infrared-dyes (IRDye800CW) were from LI-COR Biosciences. Live cell imaging 8 chamber μ-slides were obtained from Ibidi. Human embryonic kidney cells (HEK293) cells originally from the American Type Culture Collection were obtained from shared laboratory resources (Cancer Cluster, University of Saskatchewan). All other reagents were from Sigma-Aldrich unless otherwise indicated.