Promoter Luciferase Assay for Methylation

Promoter luciferase assays were performed by cloning chr6:18122895-18123314 (Supplementary Table S2 and Figure S6) into the pCpGfree-promoter-LUCIA plasmid (Invivogen, San Diego, CA, USA). In vitro M.SssI (Thermo Scientific) methylated or unmethylated pCpGfree-chr6:18122895-18123314 plasmids were co-transfected with pGL4-SV40-promoter (Promega, Madison, WI, USA) plasmid [59 (link)] into A549 and H1299 with the transfection reagent Lipofectamine 3000 (Thermo Scientific). Chemiluminescence was measured in a Tecan200pro plate reader (Tecan) in technical quadruplicates. Renilla readouts were normalized to Firefly luciferase and empty pCpGfree vector readouts.

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Faltus C., Lahnsteiner A., Barrdahl M., Assenov Y., Hüsing A., Bogatyrova O., Laplana M., Johnson T., Muley T., Meister M., Warth A., Thomas M., Plass C., Kaaks R, & Risch A. (2022). Identification of NHLRC1 as a Novel AKT Activator from a Lung Cancer Epigenome-Wide Association Study (EWAS). International Journal of Molecular Sciences, 23(18), 10699.

Publication 2022

pCpGfree-promoter-LUCIA plasmid M.SssI Lipofectamine 3000 Tecan200pro plate reader

Assays Chemiluminescence Firefly luciferase Lipofectamine Luciferase M sssi Pgl4 Plasmid Promega Renilla Sv40 Transfection Vector

Corresponding Organization : German Center for Lung Research

Other organizations : Universitat de Lleida, University Hospital Heidelberg

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Variable analysis

independent variables

Methylation status of pCpGfree-chr6:18122895-18123314 plasmid (methylated or unmethylated)

dependent variables

Chemiluminescence (luciferase activity) measured in a Tecan200pro plate reader

control variables

Co-transfection of pGL4-SV40-promoter plasmid
Use of Lipofectamine 3000 transfection reagent
Normalization of Renilla readouts to Firefly luciferase and empty pCpGfree vector readouts

controls

Positive control: Unmethylated pCpGfree-chr6:18122895-18123314 plasmid
Negative control: Empty pCpGfree vector

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