Passive lyses buffer

Using 12-well plates with C2C12 (ATCC® CRL-1772^TM) and NIH/3T3 (ATCC® CRL-1658™) cells at a density of 10⁵ cells/well, the plasmids pmiRGlo-OGN-3’UTR-wt or–Mut, and miR-155 mimic were transfected using a RNAiMAX lipofectamine (Thermo Fisher Scientific, USA). As a control, the co-transfections of anti-miR-155 or a negative control was used. The cells were transfected for 10 hours, washed with phosphate- buffered saline (PBS) 48 hours after transfection, lysed with Passive Lyses buffer (Promega, USA), and the luciferase activity was obtained using DualGlo Luciferase Assay System (Promega, USA) according the manufacturer’s instructions.

Stocks of HIV-1_NL4.3 (WT), HIV-1_NL4.3-MUT1 (MUT1), and HIV-1_NL4.3-MUT2 (MUT2) were prepared by transfecting HEK293T cells with pNL4.3 (NIH AIDS Reagent Program), pET28b-NL43-MUT1 (synthesized by ACGT Inc.) and pET28b-NL43-MUT2 (synthesized by ACGT Inc., Germantown, MD, USA) using TransIT^®-LT1 Transfection Reagent (Mirus), following the manufacturer’s protocol. Viral supernatant was collected 72 h post-transfection and filtered through a 0.45 µm filter. Viruses were titrated (TCID₅₀) by infecting TZM-bl cells (NIH AIDS Reagent Program) at multiple dilutions and incubated for 48 h. After the incubation period, cells were washed with PBS and lysed with Passive Lyses Buffer (Promega, Madison, WI, USA), followed by the addition of Luciferase Assay Reagent (Promega). Light production was measured at 560 nm absorbance. The TCID₅₀ was calculated according to the Reed and Muench method, as previously described (Reed and Muench 1938). Wells with RLU < 2.5 times background were considered negative for the calculation. Each viral stock titer was performed in triplicate, and at least two different titer determinations were performed for each batch of virus.