Miscript mirna mimic

In vitro target validation was performed using Drosophila S2 cells (Invitrogen). The cells were kept in Schneider Drosophila medium supplemented with 10% heat-inactivated FBS (Gibco) and 1% Penicillin Streptomycin (vol/vol) (Thermo Fisher Scientific) at 25 °C. Three luciferase constructs were examined: (1) a positive control containing three sites reverse complementary to miR-276 with four nucleotide linkers between each miR-276 binding site; (2) A. gambiae BCAT 3′UTR and (3) A. gambiae BCAT 3′UTR with a scrambled miR-276 binding site. All constructs were separately inserted into the multiple cloning region located downstream of the Renilla translational stop codon within the psiCheck-2 vector (Promega). psiCheck-2 reporters (100 ng) and a synthetic A. gambiae miR-276-5p miScript miRNA Mimic (100 ng, Qiagen) at a final concentration of 100 nm were co-transfected into Drosophila S2 cells using FuGENE HD transfection reagent (Promega). Cells transfected only with psiCheck-2 reporters were used as a “no miRNA mimic” control. Dual Luciferase Reporter Assay was performed 48 h post transfection using the Dual Luciferase Reporter Assay System (Promega). Firefly luciferase in the psiCheck-2 vector was used for normalization of the Renilla luciferase expression. Measurements were made in triplicates, and transfections were repeated three times independently.

MicroRNA-21 mimic (miScript miRNA Mimic, Qiagen) or miR-21 inhibitor (Antihsa-miR-21-5p; Qiagen) (Supplementary Table S1) were diluted to a final concentration of 5 nM in serum-free culture medium, including HiPerFect^® Transfection Reagent (3μL/well) (Qiagen), according to the manufacturers’ instructions, and incubated for 10 min at room temperature. Cells (NCI and FaDu) were mixed with transfected complexes, seeded at 1.5 × 10⁵ cells/well of 24-well plates and incubated for 24 h under normal growth conditions (at 37 °C and 5% CO₂). The next day, the media were removed and replaced with experimental media of 1 μM or 2 μM NNK. Untreated control groups were grown in serum-free basal media, in parallel to experimental groups. After 24 h, media were removed, the cells were washed once with PBS and total protein was isolated using M-PER reagent (mammalian protein extraction reagent; Thermo Scientific).
Assays were carried out according to the manufacturer’s instructions and performed in triplicate. All experiments were independently repeated three times.