Browning capabilities of human primary adipose-derived stromal cells compared to SGBS cells

Figures

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  Figure 1. Differentiation of SGBS preadipocytes or hASCs with indomethacin or rosiglitazone. SGBS preadipocytes (n = 5 independently performed experiments) or human adipose stromal cells (hASC, n = 7 different donors) were differentiated in vitro using either 100 µM indomethacin (Ind) or 2 µM rosiglitazone (Ro) for 14 days, representative photomicrographs shown in (A) inlets 5-fold enlarged. The adipogenic differentiation rate (B) was determined microscopically. Triglyceride content was enzymatically determined (n = 3–4) (C). The expression of key adipogenic marker genes was assessed by qRT-PCR using the dCt method, TF2B was used as reference gene (D). Mean +SEM is shown, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Figure 2. Expression of BAT-associated genes in SGBS or hASCs differentiated with indomethacin or rosiglitazone. SGBS preadipocytes (n = 5 independently performed experiments) or human adipose stromal cells (hASC, n = 7 different donors) were differentiated in vitro using either 100 µM indomethacin (indo) or 2 µM rosiglitazone (rosi) for 14 days. Total RNA was isolated and the expression of UCP1, CPT1B, PGC1a and DIO2 was determined by qRT-PCR using TF2B as reference (A). Data was also normalized using the geometric mean of adipogenic marker genes in Fig. 1D as reference (B). Mean +SEM is shown, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Figure 3. Mitochondrial activity of SGBS and hASC adipocytes. SGBS and hASCs (n = 4 each) were differentiated in vitro using either 100 µM indomethacin (Ind) or 2 µM rosiglitazone (Ro) for 14 days and subjected to respiration measurements using a plate-based respirometer (Seahorse XFe96, Agilent, A). Cells were measured in a 3 min mix – 3 min measure scheme. After 3 basal measurements, 0.5 mM dibutyryl-cAMP was injected to induce UCP1 activity by lipolysis. Oligomycin (2 µM) was added to determine ATP and proton leak-dependent respiration. Full uncoupling of mitochondrial ETC was achieved by addition of 4 µM FCCP. Finally, ETC was completely blocked by antimycin A and rotenone (1.5 µM each). Assay was performed in medium containing 1% BSA. Relative induction of respiration by cAMP was calculated as difference between first and second injection (B). Basal and proton leak respiration in cells differentiated with rosiglitazone (C) or indomethacin (D) was calculated from the oxygen consumption rate (OCR) plots. Data are shown as mean +SEM is shown, *p < 0.05, **p < 0.01.

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  Figure 4. Mitochondrial content of differentiated SGBS and hASC adipocytes. SGBS preadipocytes (n = 5) or human adipose stromal cells (hASC, n = 7) were differentiated in vitro using either indomethacin (indo) or rosiglitazone (rosi) for 14 days. Expression of mitochondrial genes were analyzed using qRT-PCR (A) and on protein level (B). Activity of citrate synthase in cell lysates (n = 3 each) was assayed as a marker for mitochondrial content (C). Mean +SEM is shown, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Figure 5. UCP1 expression in SGBS adipocytes compared to differentiated adipocytes derived from deep neck tissue. SGBS preadipocytes (n = 11) and hASCs from human deep neck (dn, n = 12) and subcutaneous neck (sc, n = 12) and mammary adipose tissue (n = 7) were differentiated into adipocytes as outlined in Fig. 1. UCP1 expression was analyzed by qRT-PCR using SDHA as reference gene. Mean +SEM is shown, *p < 0.05.