Pta luc

Luciferase assays were performed by using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The luciferase reporter and pCDH-miR-485 were cotransfected into HEK293T cells using Lipofectamine 2000. After 36 h, transfected cells were lysed in passive lysis buffer for 15 min with shaking by Rockers. Firefly luciferase activity in cell lysates was measured on a VICTOR X5 Multilabel Plate Reader (PerkinElmer, Cetus, Norwalk, CA, USA) and normalized to Renilla luciferase.
Pathway reporter vector pSRE-TA-luc was purchased from Beyotime. The negative control vectors pTA-luc and the JAK/STAT pathway reporter vector pISRE-TA-luc were obtained from Clonetech (Mountain View, CA, USA). The Wnt signaling pathway reporter vector pSuperTOPFlash luciferase reporter was constructed by inserting seven copies of the TCF/LEF binding site (AGATCAAAGG) into the pTA-luc vector, The pRL-SV40 (Promega) was used to normalized to Renilla luciferase. they were cotransfected with vectors like pcDNA3.1-Abhd2 or mimic of miR-485-5p into F9 ECs by using Lipofectamine 2000 for 36 h, Luciferase activity was detected as above.

NCI-N87 and NCI-N87R cells were transfected with pTA-Luc and TCF/LEF luciferase reporter vectors (Promega, Madison, WI, USA). We co-transfected NCI-N87 and NCI-N87R cells with the TopFlash firefly luciferase reporter vector and pRL-SV40-Renilla luciferase vector (Promega). Additionally, we incubated the cells for 72 h to detect the Wnt signaling pathway activity. The dual-luciferase reporter method (Promega) was used to determine the relative luciferase activity.