Glomax 20 20 single chamber luminometer

Luciferase assays were conducted as described previously [22 (link)]. Briefly, approximately 2.5 x 10⁴ cells were seeded per well in a 24-well plate. Transfections were conducted using Lipofectamine 2000 following manufacturer’s guidelines. Each reaction contained 50 ng of the luciferase reporter plasmid, and 2 ng pLRL-SV40 Renilla, which served as a transfection control. Where indicated, 50 ng of pcDNA3.1-β-catenin S45F [28 (link)], 50 ng of pME18 Lef [28 (link)], and 50 ng of pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro were added to the transfection. The phU6-sgRNA plasmids encoding the guide RNAs (25 ng each) were included as indicated. Total concentration of DNA was adjusted to 2 μg per reaction using pBluescript (Stratagene). Transfection mixtures were incubated on cells for 6 h, after which the media was replaced with normal growth media. Each reaction was conducted in quadruplicate. After 24 h, cells were lysed in 200 μl passive lysis buffer and luciferase levels were measured using the dual luciferase assay kit (30005–2; Biotium) on a Glomax 20/20 single chamber luminometer (Promega).

Luciferase assays were conducted as previously described [26 (link)]. Cells were seeded in quadruplicate in a 24-well plate (5 × 10⁴ cells per well) and transfected with Lipofectamine 2000. Each reaction contained 100 ng of the luciferase reporter plasmid and 2 ng pLRL-SV40 Renilla, serving as a transfection control. Total concentration of DNA was adjusted to 500 ng per reaction using pBluescript II SK (-). Where indicated, 50 ng of pcDNA3.1-β-catenin S45F and 50 ng of pME18-Lef1 were added to the transfection reaction. Transfection media were replaced after 6 h. For Dox-induced TCF7L1 or active Wnt signaling experiments, transfection media were replaced with Dox-treated media and/or Wnt3a-conditioned media (L-Wnt3a, ATCC, CRL-2647, Gathersburg, MD, USA), respectively. After 24 h, cells were lysed in passive lysis buffer (Biotium, #99821, Fremont, CA, USA) and luciferase levels were measured using the dual luciferase single tube assay kit (Biotium, #30081) on a Glomax 20/20 single chamber luminometer (Promega, Madison, WI, USA).