Figure 2

3T3-L1 adipocytes differentiated to matured white and beige type adipocytes. (A) Differentiation protocol of 3T3-L1 fibroblasts to white adipocytes and beige adipocytes. Two days after reaching confluence (day 0), cells were cultured for 2 days in medium with Dex (0.25 µM), IBMX (0.5 mM) and Ins (10 µg/ml) and then cultured in regular medium (for white adipocytes) or beige induction medium containing T₃ (50 nM), Rosi (1 µM), and IBMX (0.5 mM) (for beige-type adipocytes). (B) Representative Oil Red O staining images of differentiated 3T3-L1 white and beige adipocytes. Magnification, 100x. (C) Densitometric analysis of mitochondrial proteins in 3T3-L1 cells differentiated to matured white and beige type cells harvested at day 8 after isoproterenol (10 µM) treatment for 8 h. HSP70 served as normalization control. Western blots are shown above the columns. n = 6. (D) Ucp1 mRNA expression in 3T3-L1 white and beige adipocytes treated with 10 µM isoproterenol on day 8 for 6 h. Levels were normalized with 36B4 mRNA. n = 4. (E) miR-494-3p expression during differentiation of 3T3-L1 fibroblasts to matured 3T3-L1 white and beige adipocytes, normalized by U6. n = 3–6. (F) 3T3-L1 white and beige adipocytes expressing mito-GFP were treated with iso (10 µM) for 8 h on day 8 and stained with Mitotracker red. Magnification, 200x. Scale bars, 50 µm. (G) Quantification of the merge area (yellow) of GFP-mito (green) and Mitotracker-red (red) and adipocyte numbers (quantified by DAPI). n = 4. (H) miR-494-3p expression in 3T3-L1 beige cells with or without 10 µM isoproterenol for 6 h, normalized by U6. n = 3. (I) Western blot of mitochondrial proteins during differentiation of 3T3-L1 cells to matured white and beige adipocytes. Pan-actin served as loading control. (J) Oxygen consumption rate (OCR) in 3T3-L1 white and beige cells pre-treated with H89 (50 µM) for 1 h with the sequential injection of isoproterenol (50 µM), oligomycin (2 µM), FCCP (2 µM), rotenone (R; 0.5 µM) and antimycin A (AA: 0.5 µM) at the indicated times. *p < 0.05, **p < 0.01. Full-length blots are presented in Supplementary Fig. 5. Ucp1: uncoupling protein 1; PGC1-α: PPAR gamma coactivator 1-α; TFAM: transcription factor A mitochondria; PDH: pyruvate dehydrogenase E1-alpha subunit; MTCO1: cytochrome c oxidase subunit 1; SDHA: succinate dehydrogenase subunit A; ANT1/2: adenine nucleotide translocase 1/2; HSP70: heat shock protein 70; P-Actin: pan-actin; iso: isoproterenol; Oligo: oligomycin; RAA: rotenone and antimycin A.