Additional supporting information can be found in the online version of this article.

ANA_22275_sm_suppinfoFig1.eps2271KSupplementary Fig 1 V5 epitope tagged DUX4 caused apoptosis in vitro. (A) To simplify DUX4 protein detection, we first added a C-terminal V5-epitope tag to normal human DUX4 cDNA by PCR and cloned the resultant product into a mammalian expression vector used in all cell and mouse experiments. ITR, AAV inverted terminal repeat; CMV, cytomegalovirus promoter; pA, SV40 poly A signal. (B) Following transfection of expression plasmids into HEK293 cells, we confirmed DUX4 protein expression by western blot using V5-epitope antibodies. (C) DUX4 was previously shown to induce apoptosis in vitro. We validated that the V5 tag did not impact DUX4 pro-apoptotic function by measuring Caspase 3/7 activity in HEK293 cells transfected with plasmid expressing DUX4.V5, untagged DUX4, FRG1, and mock or empty vector controls. *, indicates significantly increased Caspase 3/7 activity compared to mock lipofectamine or empty CMV vector (pCINeo) controls, or CMV.FRG1, p<0.0007 (ANOVA).
ANA_22275_sm_suppinfoFig2.eps5117KSupplementary Fig 2. (A) Real-time PCR shows DUX4 and DUX4.HOX1 were expressed at equivalent levels in vivo. N=4 muscles/group. Differences are not statistically significant (ANOVA). (B) Representative western blots showing DUX4 and DUX4.HOX1 proteins were expressed at equivalent levels in vivo. Each lane contains muscle protein extracts harvested 10 days post-injection from low dose AAV.DUX4.HOX1 or AAV.DUX4-injected animals. Numbers 516, 517, 518, 153, 520, 521 indicate individual mouse identification numbers. HEK293 DUX4 lane contains protein extract harvested from HEK293 cells transfected with a CMV.DUX4V5 expression plasmid, for use as a positive control. Blots were incubated with anti-V5 or anti-DUX4 primary antibodies. Multiple DUX4 and DUX4.HOX1 products were evident, but preodominant bands migrated at 50 kDa and ∼38 kDa, both of which were previously reported DUX4 protein isoforms2,4. The smaller sections in this panel are cropped around the 50 kDa marker and digitally enhanced to better show products corresponding to full-length DUX4 protein size. The DUX4-specific antibody consistently produced weaker bands compared to the V5 antibody. The bottom panel shows the membrane stained with Napthol Blue Black, to demonstrate equal protein loading.
ANA_22275_sm_suppinfoFig3.eps26634KSupplementary Fig 3. DUX4-transduced myofibers are TUNEL-positive. (A) Low power photomicrographs demonstrating co-localization of TUNEL-positive nuclei in DUX4-, but not DUX4.HOX1- transduced myofibers. DUX4 was localized in predominantly in nuclei, but also in the cytoplasm of degenerating myofibers. Arrows indicate nuclear-localized DUX4 present within normal myofibers. Caret indicates DUX4+/TUNEL+ myonuclei present within overtly normal myofibers. Asterisk specifies TUNEL-negative degenerating myofibers containing cytoplasmic DUX4. Arrowheads point to DUX4+/TUNEL+ degenerating myofibers. (B) High power image of DUX4+/TUNEL+ degenerating myofiber in (A). Scale bar, 50 μm.
ANA_22275_sm_suppinfoFig4.eps23346KSupplementary Fig 4. DUX4-transduced myofibers express cleaved Caspase-3. (A-D) show low power H&E, caspase-3 (green), DUX4 (red), and DAPI (blue) stained serial sections from DUX4-transduced mouse muscle 10 days post-injection. Caret, asterisk, and pound sign serves as landmarks for orientation of high power images in panels E-L. (E-F) shows nuclear DUX4 in histologically normal myofiber (caret). (G-H) shows Caspase-3 positive myofibers containing nuclear-localized DUX4 (pound sign). (I-J) Adjacent to landmarked myofibers (indicated by asterisk and pound sign) are examples of Caspase-3 positive myofibers containing nuclear- and cytoplasm-localized DUX4. Some mononuclear cells may also be caspase-3 positive. Antibody used in this figure specifically detected cleaved caspase-3 (9662, Cell Signaling Technology). Scale bars, 50 μm.
ANA_22275_sm_suppinfoTable.doc155KSupplementary Table. Total apoptotic gene expression changes in DUX4-injected muscles
Supplementary_Methods_and_Figure_Legends.doc52KSupplementary Methods.

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