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

Figure 2.

Figure 2

Contribution of CpG/GC-rich and repetitive nature to copy-number dependent repression. A-C. Schematic view of constructs named: 12 DUX4p (A), CGI B3gnt6 (B), 11-, 5-, 1-HbG (C). The constructs carry: 11 copies of DUX4p repeats (overall, 110 CpG and 72% GC, A); a non-repetitive sequence containing a CpG island (CGI) from B3gnt6 locus (110 CpG and 60% GC, B); 11, 5 or 1 copies of HbG repeats (each with 0 CpG and 38% GC, C). Additionally, all constructs retain a DUX4p unit prolonged to include DUX4 ATG sequence, and an EGFP reporter gene (A-C). Upon site-specific integration of the constructs in C2C12-frt muscle cells, the isogenic cells (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. 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 five (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.