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. 2020 Nov 24;8:586179. doi: 10.3389/fcell.2020.586179

FIGURE 1.

FIGURE 1

Upregulation of NAD+ biosynthesis in the salvage pathway is required for 3T3-L1 preadipocyte differentiation. (A) Schematic of NAD synthesis in the salvage pathway. (B) Intracellular levels of NMN and NAD+ were measured with LC-MS during the differentiation of 3T3-L1 cells (n = 3). Data are represented as mean ± SD. (C) Relative expression levels of enzymes in the NAD+ synthetic pathway during the differentiation of 3T3-L1 cells (n = 3). Data are represented as mean ± SD. (D) Western blotting for enzymes in the NAD+ synthetic pathway during the differentiation of 3T3-L1 cells. The representative image from three independent experiments. The signals of Nampt and Nmnat1 were quantified and adjusted with those of β-actin (n = 3). (E) 3T3-L1 cells were treated with 100 nM FK866 or 100 nM FK866 and 100 μM NMN during differentiation. Intracellular levels of NMN and NAD+ were measured on day 4 after inducing differentiation (n = 4). Data are represented as mean ± SD. (F) Oil Red-O staining of differentiated 3T3-L1 cells treated with 100 nM FK866 or 100 nM FK866 and 100 μM NMN on day 8 after inducing differentiation. The lipid accumulation was quantified as absorbance. (n = 4) Ctrl represents control. *p < 0.05, **p < 0.01.