Tcf reporter plasmid

The fragment of 3′UTR of GSK3β containing the binding site of miR-1290 was inserted into the pGL6-miR‐based luciferase reporter vector. Next, PC3 cells were co-transfected with well-designed pGL6-miR‐based reporter plasmids, along with a miR-1290 agomir using Lipofectamine 2000 for 48 h. Subsequently, the Dual Luciferase Reporter Assay System (Beyotime) was used to measure luciferase activity in cell lysates. For the TOPflash reporter assay, PC3 cells were co-transfected with TOPflash reporter gene (TCF Reporter Plasmid; Millipore), along with anti-miR-1290. At 48 h post-transfection, the reporter activities were assayed by Dual Luciferase Reporter Assay System (Beyotime). Renilla luciferase activity was used for normalization.

XTT assays were performed according to the manufacturer's instructions (Biotium). In short, HCT116, DLD-1, HT-29 or SW480 cells were plated at 2–4 × 10³ cells per well (n=8 per condition) in 96-well plates (Corning) and XTT assays were performed on five consecutive days. For TOPflash luciferase reporter assays, HCT116 or SW480 cells were co-transfected with the TCF Reporter Plasmid (Millipore) and pcDNA3.1(+) vector (Thermo Scientific) that contains a Geneticin-selectable marker. Stable lines were selected with 800 μg ml⁻¹ Geneticin (Thermo). The cells were lysed in total cell lysis buffer and Luciferase substrate (Promega) was added, followed by measurement of bioluminescence.

Cells were transiently transfected with TOPflash reporter gene (TCF reporter plasmid, 21-170, Millipore) and Renilla-Luc as an internal control, respectively. Cells were harvested 48 h after transfection in passive lysis buffer (Promega) and assayed by the Dual Luciferase Reporter Assay System (Promega) using GloMax luminometer (Promega). Renilla luciferase activity was used for normalization.