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TOP/FOP-Flash luciferase reporter analysis

SL Shihai Liu
JQ Jing Qiu
GH Guifang He
WH Weitai He
CL Changchang Liu
DC Duo Cai
HP Huazheng Pan
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Wnt/β-catenin signaling pathway activity was quantified via a TOP/FOP-Flash luciferase reporter assay, with the pRL-SV40 vector being used as an internal control. Control and NC cells were co-transfected with pRL-SV40 and TOP/FOP flash (Promega, WI, USA). Cells in all groups were co-transfected with pRL-SV40 and TOP/FOP flash, and then treated them for 24 h with LiCl (20 mM), with those in the TRAIL + IER3 group additionally being infected with the TRAIL and IER3-encoding adenoviruses. Luciferase activity was then quantified using a Dual-Luciferase Reporter Assay System (Promega, WI, USA), with the TOP/FOP ratio being used to quantify Wnt/β-catenin signaling pathway activity following normalization of firefly luciferase activity to Renilla luciferase activity.

we transfected Huh7 cells with the appropriate reporters, and then treated them for 24 h with LiCl (20 mM), after which cells were infected with Ad5-TRAIL and Ad5-IER3 or Ad5-EGFP. LiCl treatment was confirmed to increase the TOP/FOP ratio of these HCC cells (P < 0.01), consistent with Wnt/β-catenin signaling pathway activation.

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