Beta glo luminescent assay kit

NP doses were selected on the basis of our previous findings [6] , and in vitro growth inhibition assays were performed as previously described [6] . Briefly, purified parasite suspension plus the NPs (reconstituted in culture medium prior to use) was added to growing HFF monolayers and incubated for 48 h. The untreated but infected cells served as controls, whereas the culture medium only well was used to correct for the background signal. Sulfadiazine (Sigma, St Louis, MO, USA) and/or pyrimethamine (Wako Pure Chemical, Osaka, Japan) were included as positive controls. After the 48-h incubation at 37 °C in a 5% CO₂ atmosphere, the viability of the RH-2F parasite strain was determined by assaying for galactosidase activity by using a Beta-Glo luminescent assay kit (Promega, Madison, WI, USA). The assay was performed in triplicate and repeated three times independently. All experiments were performed in 96-well solid white plates (Nunc; Fisher Scientific, Pittsburgh, PA, USA) unless otherwise stated.

For the validation of Tbx3 as a direct target of miR-206, wild-type and mutant pMIR-REPORT luciferase vectors were purchased from Applied Biological Materials Inc. (ABM MT-h25249). The miR-206 wild-type binding sequence or its mutated form was inserted at the C terminus of the luciferase gene to generate pMIR-TBX3-3′ UTR (containing the full-length 3′ UTR segment of human Tbx3) or pMIR-TBX3-mut-3′ UTR (deleting five nucleotides in the seed region), respectively. Briefly, HEK-293 T (2.5 × 10⁴) cells were seeded into 24-well plates, cultured overnight in antibiotic-free media and co-transfected with 250 ng of pMIR-TBX3-3′ UTR, pMIR-TBX3-mut-3′ UTR or control plasmids, with 50 nM miR scrambled control (NC) or 50 nM miR-206 mimic using Lipofectamine 2000. Forty-eight hours post transfection cells were collected in reporter lysis buffer (Promega) and luciferase activity was measured using Dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's protocol, and detected by Glomax 96 microplate luminometer (Promega). β-galactosidase activity was used for normalisation, and measured by beta-glo Luminescent Assay Kit (Promega).