Fig. 8.

DUX4 deletion and fusion constructs that were non-toxic and had intact homeodomains were dominant-negative inhibitors of DUX4-FL. (A) For this experiment, four plasmids were co-transfected into HEK293 cells including (i) pCS2-DUX4-FL-V5, which is a positive activator of the 12X reporter, (ii) the DUX4 deletion or fusion construct that was to be tested for generation of a dominant-negative inhibitor, (iii) the 12X-DUX4 promoter-Luciferase reporter, to measure DUX4 promoter binding and subsequent activation of the luciferase reporter gene, and (iv) a Renilla luciferase reporter, to measure transfection efficiency for use in normalization. (B) As indicated, activation by DUX4-FL of the 12X promoter was inhibited only by those constructs that had two intact homeodomains and were non-toxic in other assays (see Figs. 2-7). HEK293 cells were transfected with a 1:3 ratio of DUX4-FL to test plasmid, and activity of the 12X reporter was measured 24 h after transfection. ***P<0.001 compared to pCS2-V5 control by the Dunnett method; means±s.e.; n=4.