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. 2016 Dec 28;26(4):753–767. doi: 10.1093/hmg/ddw426

Figure 1.

Figure 1

DUX4p repeats mediate copy-number dependent repression in isogenic muscle cells. A–C. Top, schematic view of constructs used for Flp-FRT-mediated recombination and carrying 1 (A), 5 (B), or 12 (C) DUX4p repeats and an EGFP reporter gene under the control of the DUX4 promoter. Upon site-specific integration in C2C12-frt muscle cells, the isogenic cells differing for DUX4p copy number (A–C) were tested for EGFP expression. For relative quantification of EGFP protein levels among samples, flow cytometry analyses of EGFP Median Fluorescence Intensity (MFI) (D) and of percentage of EGFP+ events (E) are shown. Examples of histogram analyses with EGFP (x axis) versus counts (y axis) are reported (A–C, bottom), compared to a non-fluorescent (wild type) cell line. For relative quantification of EGFP at the RNA level, RT-qPCR analyses of EGFP (F) and Gapdh negative control (G) are presented over β-Actin. Results are expressed over 1 DUX4p (D–G). The mean of the signals obtained from six (D,E) or four (F,G) independent experiments is shown. The error bars represent SEM of independent biological replicates. Asterisks indicate statistical significance (p value) as evaluated by One way ANOVA Bonferroni’s multiple comparison test. ****=P < 0.0001; **= P < 0.01.