*2.18. Microarray Analysis*

Microarray analysis was conducted with the Agilent Technologies mouse GE4X 44K (V2) chip to determine the differentially expressed genes in wild-type (TTRwt) and TTRkd (TTR knockdown) cells as described previously [30]. Briefly, TTRwt and TTRkd cells were grown in serum (+) differentiation media for two days and RNAs were extracted, synthesized into cDNA with fluorescence using a Low RNA Input Linear Amplification kit (Agilent Technologies, CA, USA) according to the manufacturer's instructions. A total of three hybridizations were performed, and the statistical relevance of gene expression differences was confirmed by SAM (Standard University, Palo Alto, CA, USA). The significance cut-off was a median false discovery rate ≤5% for the SAM analysis.

### *2.19. DAVID Analysis*

DAVID was performed as described previously [5]. In brief, enriched biological themes in up- and down-regulated gene lists (*p* ≤ 0.05 and 2 fold≤) were categorized by employing the Gene Ontology (GO) terms of cellular component, molecular function, and biological process in DAVID.

#### *2.20. Statistical Analysis*

Mean values of normalized expressions were evaluated by Tukey's Studentized range test to categorize expressional differences of genes, considering *p* ≤ 0.05 statistically significant. The real-time RT-PCR data was normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal standard and was analyzed by one-way ANOVA using PROC GLM in SAS 9.0 (SAS Institute, Cary, NC, USA).

### **3. Results**

#### *3.1. TTR Secretion During Myoblast Di*ff*erentiation*

To investigate TTR secretion from cells during C2C12 myoblast differentiation, normal and TTR knockdown cells were cultured in serum-free media for two days, after which the isolated protein level from cultured media was analyzed by Western blotting. The appearance of more TTR protein in cultured media compared to that in cell lysate indicates that TTR is secreted during myoblast differentiation (Figure 1A). Furthermore, TTR mRNA and protein were decreased in TTRkd cells and cultured media, respectively, compared to those in TTRwt cells (Figure 1A). Next, the TTR mRNA level was analyzed in normal cells and exosomes from mouse plasma and media of cultured cells (CM): T4-treated cells, TTRwt, and TTRkd. TTR mRNA was evident in exosomes isolated from culture media and mouse plasma and was increased by T4 treatment but decreased by TTRkd (Figure 1B). TTR immunoneutralization using TTR antibody was performed during differentiation. Myotube formation and the expression of the myogenic genes were decreased by TTR neutralization. However, TTR expression was significantly enhanced in neutralized cells (Figure 1C,D). Interestingly, when the T4 concentration was measured in cells, it was higher in non-neutralized cells than in neutralized cells supplemented with T4 (Figure 1E). Taken together, these results show that TTR secreted from cells transported T4 into the cells during myoblast differentiation.
