Dual light reporter system

HEK-293 or NIH-3T3 cells (12,500 cells/well) were transfected with pMSCV-IRES-eGFP plasmids encoding DUX4 and/or Pax7, or dominant negative DUX4-ERD, or dominant negative Pax7-ERD^{15 (link), 29 (link)}, together with reporter plasmids encoding either DUX4-responsive promoters driving luciferase expression (pZSCAN4-luc^{35 (link)} or RFPL4B-luc) or a Pax3/7-responsive element driving β-galactosidase (p34 plasmid^{15 (link)}) using Lipofectamin LTX (Thermofisher). A pRSV vector encoding either β-galactosidase or luciferase as appropriate was co-transfected as an internal control for normalisation of transfection efficiency. Total amount of DNA transfected per reaction was 1.5 mg, in equal ratios for reporters and plasmids encoding genes of interest, and 250 ng/reaction of the internal normaliser vector. Cells were harvested 24 h after transfection, and assayed using the Dual-light Reporter system (Thermofisher) according to the manufacturer’s instructions. 3-4 independent transfections were performed in technical triplicate. Reporter gene activity was measured using Glomax-Multi + plate reader (Promega) and normalised for transfection efficiency. N = 3/4 for each cell line, analysis of variance (ANOVA) revealed significant intensity differences, post hoc unpaired two-tailed t-tests were employed to assess significant pairwise differences.

3’ UTR luciferase experiments
B35 neural progenitor cells were plated in a 96 well plate (Celltreat Scientific Products) at a concentration of 1.0 × 10⁵ cells/mL and allowed to adhere overnight. The next day cells were co-transfected with axolotl c-Jun 3’ UTR luciferase reporter, β-Galatosidase internal control and 100 nM of miR-200a or control mimic (Qiagen) Lipofectamine 3000 (Invitrogen). After 48 h luciferase activity was determined using Dual Light Reporter system (Thermo) according to the manufacturers protocol.

C2C12 myoblasts were co-transfected using Lipofectamine LTX (Thermofisher) with DUX4c or DUX4 constructs or control GFP and DUX4-responsive promoters driving luciferase (pZSCAN4-luc, pKHDC1L-luc, RFPL4b-luc), together with a pRSV-β-galactosidase construct to normalise transfections. Myoblasts were harvested 24 h later, and assayed using the Dual-light Reporter system (Thermofisher) in three transfections measured in triplicate on a Glomax-Multi+ plate reader (Promega).