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. 2023 Oct 5;9(10):e20713. doi: 10.1016/j.heliyon.2023.e20713

Fig. 1.

Fig. 1

Determination of an optimal protocol for inducing adipogenic differentiation of 3T3L-1 preadipocytes by assessing adipogenesis-related gene expression and intracellular metabolic function in 2D culture.

Three different protocols for inducing adipogenic differentiation in 2D cultured 3T3-L1 cells were investigated: 1) addition of 250 nM dexamethasone during the initial two days, and addition of 500 μM IBMX and 1 μg/ml insulin each day for 7 or 10 days (=IBMX), 2) addition of 250 nM dexamethasone and 10 nM T3 during the initial two days, and addition of 10 μM troglitazone and 1 μg/ml insulin each day for 7 or 10 days (=Tro + T3), and 3) addition of 250 nM dexamethasone during the initial two days, and addition of 500 μM IBMX, 10 μM troglitazone, and 1 μg/ml insulin each day for 7 or 10 days (=IBMX + Tro). Each sample was subjected to quantitative RT-PCR analysis for adipogenesis-related genes (n = 3) and cellular metabolic function analyses using a Seahorse XFe96 Bioanalyzer (n = 5). Panel A: Expression levels of mRNA of Pparγ, Cebpa, Fabp4, Adiponectin, and Leptin in the three different protocols. Panel B: Measurement of the rate of oxygen consumption (OCR). Panel C: Measurement of the rate of extracellular acidification (ECAR). Panel D: Levels of major indices of mitochondrial functions and glycolytic functions. Basal OCR was determined as the average of OCR at baseline minus the average of OCR after a rotenone/antimycin injection. Maximal respiration was determined as the average of OCR after FCCP injection minus the average of OCR after a rotenone/antimycin injection. Basal ECAR was determined as the average of ECAR at baseline minus the final measurement of ECAR after 2-DG injection. Glycolytic capacity was determined as the average of ECAR after an oligomycin injection minus the final measurement of ECAR after injection of 2-DG. Data are presented as means ± the standard error of the mean (SEM). *P < 0.05, (ANOVA followed by Tukey's multiple comparison test). Tro: troglitazone, Oligo: oligomycin, FCCP: carbonyl cyanide p-trifluoromethoxyphenylhydrazone, R/A: rotenone/antimycin A, 2DG: 2-deoxyglucose.