Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts

• Published in: Molecules (Basel, Switzerland) Vol. 20; no. 5; pp. 8304 - 8315
• Main Authors: Pandey, Sachchida Nand, Khawaja, Hunain, Chen, Yi-Wen
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 08.05.2015; MDPI
• Subjects: Adult; Cell Culture Techniques - methods; Cells, Cultured; Cytotoxicity; dexamethasone; Dexamethasone - pharmacology; DNA Primers - genetics; DNA, Complementary - genetics; DUX4; FSHD; Homeodomain Proteins - biosynthesis; Homeodomain Proteins - genetics; Humans; KOSR; Male; Middle Aged; Muscle, Skeletal - metabolism; Muscular dystrophy; Muscular Dystrophy, Facioscapulohumeral - metabolism; Muscular Dystrophy, Facioscapulohumeral - pathology; Myoblasts - metabolism; RNA-Directed DNA Polymerase - metabolism; Superscript; United States > US; KOSR; dexamethasone; FSHD; Superscript; DUX4
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules20058304

Facioscapulohumeral muscular dystrophy (FSHD) is believed to be caused by aberrant expression of double homeobox 4 (DUX4) due to epigenetic changes of the D4Z4 region at chromosome 4q35. Detecting DUX4 is challenging due to its stochastic expression pattern and low transcription level. In this study, we examined different cDNA synthesis strategies and the sensitivity for DUX4 detection. In addition, we investigated the effects of dexamethasone and knockout serum replacement (KOSR) on DUX4 expression in culture. Our data showed that DUX4 was consistently detected in cDNA samples synthesized using Superscript III. The sensitivity of DUX4 detection was higher in the samples synthesized using oligo(dT) primers compared to random hexamers. Adding dexamethasone to the culture media significantly suppressed DUX4 expression in immortalized (1.3 fold, p < 0.01) and primary (4.7 fold, p < 0.01) FSHD myoblasts, respectively. Culture medium with KOSR increased DUX4 expression and the response is concentration dependent. The findings suggest that detection strategies and culture conditions should be carefully considered when studying DUX4 in cultured cells.