Figure 5
miR-494-3p overexpression or inhibition in primary adipocytes. (A) Stromal vascular (SV) cell fractions were isolated from iWAT and differentiated to beige adipocytes using the indicated protocol as described in Methods. Cells were treated with dexamethasone (0.25 µM), IBMX (0.5 mM), insulin (10 µg/mL), T3 (1 nM), and rosiglitazone (0.5 µM). (B) Ucp1 expression in beige primary adipocytes with or without isoproterenol treatment (50 µM, 8 h). Results are representative of three independent experiments. Pan-actin served as loading control. (C) Densitometric analysis of results from (B). Pan-actin served as normalization control. n = 3. (D) miR-494-3p expression in primary beige adipocytes with or without isoproterenol (50 µM, 8 h). Levels were normalized using U6. n = 3. (E,F) miR-494-3p and miR-494-5p expression during differentiation of primary beige adipocytes from iWAT at different time points. Levels were normalized to U6 as an internal control. n = 4. (G) mtDNA copy number was analysed in cells harvested at day 8 after iso (10 µM) treatment for 8 h as described in Method (supplementary file 1). (H,J) Western blot of in primary adipocytes with miR-494-3p overexpression (H) or inhibition (J). Cells were harvested at day 8 with iso (10 µM) treatment for 8 h. Results are representative of three independent experiments. n = 6. HSP70 served as an internal control. (I,K) Densitometric analysis of western blot results (H) and (J). HSP70 served as control. n = 6. *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; ANT1/2: adenine nucleotide translocase 1/2; MTCO1: cytochrome c oxidase subunit 1; HSP70: heat shock protein 70; iso: isoproterenol.
