Lipofectamine 2000 transfection reagent

Non-viral piggy-bac vectors (1–2 μg) were co-transfected with mouse transposase-expression vector (0.5 μg) in SCA1+ cells, MEFs, and MSCs, using Lipofectamine 2000 Transfection Reagent (11668-027, Sigma). MirVana miRNA inhibitor (Applied Biosystems, Carlsbad, CA, USA) was used to knockdown mir-300 expression in a separate set of experiments. Transfection studies, in parallel with negative control miRNA inhibitor experiments, were carried out with lipofectamine RNAiMax Transfection Reagent (13778030-Sigma).

Human BC cell line HT-1197 (ATCC, USA) was used. Eagle’s Minimum Essential Medium was mixed with 10% fetal bovine serum (FBS) and was used as the culture medium. Cells were cultivated at 37 °C with 5% CO₂. PLAC2 and TGF-β1 expression vectors were constructed using pcDNA3.1 vector (Sangon, Shanghai, China). Negative control miRNA and miR-663 mimic were purchased from Sigma-Aldrich (USA). Inhibitor negative control and miR-663 inhibitor were also purchased from Sigma-Aldrich (USA). HT-1197 cells were harvested at the confluence of 70–90%. Next, 10 nM of LAC2 and TGF-β1 expression vector, 10 nM of empty pcDNA3.1 vector (negative control, NC), 30 nM of negative control (NC) miRNA and miR-663 mimic, or 30 nM of inhibitor negative control (NC) and miR-663 inhibitor were transfected into 10⁵ cells using lipofectamine 2000 transfection reagent (Sigma-Aldrich, USA). Cells without transfections were used as the control (C). the following experiments were performed using cells collected at 24 h post-transfection.

Cell lines SCC25 and CAL27 were transfected with the special small interfering RNA (siRNA, CCT3, 5′-UGAAAGUAAAGUAUUCAUCUCGAUGAAUACUUUACUUUCAUC-3′;) or a non-specific control using Lipofectamine 2000 Transfection Reagent (Sigma-Aldrich) according to the manufacturer’s instructions.