## 2 Results  
### Characterization of Adipogenic Differentiation and SAMM50 Expression of Beige Adipocytes  
The human adipose-derived stem cells (hADSCs) were differentiated into white and beige adipocytes by treating them with an adipogenic cocktail for 12 days (Figure 1A). Oil Red O (ORO) staining showed significantly increased lipid accumulation in the differentiated groups than in the hADSC group (Figure 1B, \(p<0.005\)). Additionally, the expression levels of adipogenic markers were significantly elevated in the beige and white groups compared with the hADSC group (Figure 1C, \(p<0.05\)). To determine whether beige adipocytes were properly differentiated, a beige adipogenic marker, _UCP1_, was investigated, and it was highly expressed in the beige group compared to the hADSC group and the white group (Figure 2A and Figure S1, \(p<0.005\)). Thermogenic markers, including _PPARGCIA_, _DIO2_, _ELOV1_, _CIDEA_, and _CIDEC_ were expressed significantly higher in the beige group compared with the hADSC group and the white group (Figure 2B, \(p<0.005\)). Moreover, the expression levels of beige-specific markers, including _PAT2_, _SLC25A20_, _FABP3_, _PDK4_, and _CITED1_, were also higher in the beige group compared to the hADSC and white groups (Figure 2C, \(p<0.005\)). In particular, the SAMM50 mRNA and protein levels were significantly higher in the beige group compared with both the hADSC and white groups (Figure 2D,E, \(p<0.005\)). Figure 1: The adipogenic differentiation of human adipose-derived stem cells. (**A**) Schematic images of human adipose-derived stem cell differentiation into white and beige adipocytes. (**B**) The cells were stained with Oil Red O. The lipid accumulation levels were measured by absorbance at 490 nm by extracting Oil Red O dye with isopropanol. (**C**) The expression levels of adipogenic genes were assayed by qPCR. Results are shown as mean \(\pm\) SEM. \({}^{*}\)\(p<0.05\); \({}^{**}\)\(p<0.01\); \({}^{***}\)\(p<0.005\) compared to the hADSC group. The Expression of Mitochondrial Dynamics Genes Is Regulated by Modulating the Expression of SAMM50 in Beige Adipocytes  
The regulatory action of SAMM50 in the beige adipocyte's physiology was analyzed using the short hairpin RNA (shRNA) and expression vectors. Firstly, the knockdown of SAMM50 inhibited both the mRNA and protein levels of SAMM50 in the infected hADSCs. Especially, the expression of SAMM50 was significantly suppressed in the shSAMM50-2 group compared to the NT group (Figure 3A,B, \(p<0.01\)). Meanwhile, the FLAG-tagged SAMM50, which was infected in hADSCs with the use of a lentivirusirus, significantly increased the SAMM50 mRNA and protein levels in the FLAG group (Figure 3C,D, \(p<0.05\)). Additionally, the protein level of FLAG was evaluated, confirming the overexpression of FLAG protein in the FLAG-SAMM50 group (Figure S2, \(p<0.01\)). In order to identify the role of SAMM50 in the mitochondria of beige adipocytes, the expression levels of the mitochondrial dynamics-related genes were analyzed. These results showed that the expression levels of the mitochondrial fusion markers, including _MFN1_, _MFN2_, and _OPA1_ were significantly down-regulated in the shSAMM50-2 group compared with the NT group (\(p<0.005\)), however, there was no significant difference shown in the FLAG-SAMM50  
Figure 2: The establishment of beige adipocytes differentiated from human adipose-derived stem cells and SAMM50 expression in beige adipocytes. (**A**) qPCR analysis assessed _UCP1_ mRNA levels in the human adipose-derived stem cells and differentiated cells. (**B**,**C**) The expression levels of thermogenic (**B**) and beige-specific (**C**) genes were investigated by qPCR. (**D**,**E**) qPCR and Western blot analyses were performed to examine mRNA and protein levels of SAMM50 in the human adipose-derived stem cells and the differentiated cells. The protein levels of SAMM50 were quantified using ImageJ software. Results are shown as mean \(\pm\) SEM. ***\(p<0.005\) compared to the hADSC and white group.