Transcriptional Regulation by DUX4 and Pax7

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.