A Targeted Approach for Evaluating DUX4-Regulated Proteins as Potential Serum Biomarkers for Facioscapulohumeral Muscular Dystrophy Using Immunoassay Proteomics

Figures

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  Fig. 1. DUX4-induced ALPP protein is present in muscle cell supernatant. (A) Venn diagram showing the overlap between unique protein targets on the Olink Proteomics human Target 96 panels and DUX4-induced genes as classified in [31]. (B) The protein level of ALPP (left), CA2 (middle), and CRHBP (right) as measured by Olink Proteomics assay in cell lysate (top) and supernatant (bottom) harvested from MB135-iDUX4 myoblasts left untreated (-DOX) or treated with doxycycline (+DOX) to induce DUX4 transgene expression for 12 or 24 hours. (C) ALPP protein levels as measured by Olink Proteomics assay in cell lysate (top) and supernatant (bottom) from MB135-iDUX4 myoblasts treated with or without doxycycline for 24 hours following transfection with no (-), non-targeting control (siCTRL), or DUX4 (siDUX4) siRNA. Protein levels in (B) and (C) are presented as normalized log2 expression values. LOD, limit of detection. Each data point in the plots represents a single replicate; replicates from comparable conditions in (B) and (C) are grouped together in Supplementary Figure 1C.

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  Fig. 2. ALPP protein levels increase upon FSHD myoblast differentiation. (A) The normalized log2 expression level of ALPP protein as measured by Olink Proteomics assay in the cell lysate (left) and supernatant (right) of two independent control and three independent FSHD myoblast cell lines differentiated into myotubes for 2, 4, or 6 days. (B) DUX4 target gene ZSCAN4 mRNA levels as measured by RT-qPCR in control and FSHD myoblast cell lines differentiated into myotubes for 0, 2, 4, or 6 days. LOD, limit of detection. n/a, not available.

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  Fig. 3. Serum ALPP levels do not predict FSHD disease state. (A) ALPP protein levels presented as normalized log2 expression values measured by Olink Proteomics assay in serum from 20 individuals with FSHD and 20 unaffected controls. (B–D) ALPP protein levels shown in (A) stratified by sex (B), age (C), and FSHD Clinical Severity Score (D).