Fig 2: Iron deficiency-induced myostatin regulation by DAX1. (a) Expression of myostatin mRNA in C2C12 myotubes transfected with a nuclear receptor for 48 h. AR: androgen receptor; CAR: constitutive androstane receptor; DAX1: dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region, on chromosome X, gene 1; ERa: estrogen receptor a; ERRa/ß/?: estrogen-related receptor a/ß/?; FXR: farnesoid X receptor; GR: glucocorticoid receptor; HNF4a: hepatocyte nuclear factor 4 a; LRH-1: liver receptor homolog-1; LXRa: liver X receptor a; PXR: pregnane X receptor; RORa: RAR-related orphan receptor a; SF-1: steroidogenic factor 1; SHP: small heterodimer partner; THRa: thyroid hormone receptor a; TR4: testicular receptor 4. (b) Expression of DAX1 mRNA in C2C12 myotubes treated with different doses of deferoxamine (DFO) and FeSO4. (c) Expression of DAX1 mRNA in C2C12 myotubes with DFO in the presence or absence of FeSO4. (d) Adenoviral overexpression of DAX1 and mRNA expression of myogenic and muscle atrophy-associated factors. C2C12 myotubes were infected with an adenovirus expressing DAX1 (Ad-DAX 1) or control vector (Ad-CTL) at 100 multiplicity of infection for 48 h. Myf5: myogenic factor 5; MyoD: myoblast determination protein 1; MuRF1: muscle RING-finger protein-1. (e) Protein expression of muscle atrophy-associated factors in C2C12 myotubes infected with Ad-DAX 1. GAPDH: glyceraldehyde 3-phosphate dehydrogenase. A densitometric analysis of the Western blots is shown in the graph on the right. Data represent mean ± SEM. * p < 0.05.

Fig 3: Protein levels of myogenic regulatory factors (MRFs). The protein levels of MyoD, Pax7, Myf5, Myf6 and MyBPH were analysed by western blotting using the longissimus dorsi muscles of 1-day-old piglets and adult pigs of Berkshire and JNPs (a). Protein levels were normalized to ß-actin, and relative intensity of MyoD (b), Pax7 (c), Myf5 (d), Myf6 (e) and MyBPH (f) are represented. Values are presented as mean ± standard error of mean (s.e.m.) (*p < .05, **p < .01 and ***p < .001; two-tailed t-test). JNP: Jeju native pig

Fig 4: Transcription levels of MRFs. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis was performed in Berkshire and Jeju native pigs (JNP). The expression levels of MyoD (a), Pax7 (b), Myf5 (c), Myf6 (d) and MyBPH (e), normalized against ß-actin were represented. Values were presented as mean ± standard error of mean (s.e.m.) (*p < .05 and **p < .01; two-tailed t-test). JNP: Jeju native pig