Adiponectin

qPCR was performed on a LightCycler 2.0 instrument with the LightCycler FastStart DNA Master^PLUS SYBR Green I kit (Roche Diagnostics, Mannheim, Germany). The mRNA levels of the genes of interest were first normalized to HPRT (hypoxanthine-guanine-phosphoribosyltransferase, ΔCT value) and then to the respective control condition (ΔΔCT value). The ΔΔCT value was used for the determination of the relative expression. The following oligonucleotide primers were obtained from ThermoScientific (Ulm, Germany): adiponectin forw 5′-GGC CGT GAT GGC AGA GAT-3′, adiponectin rev 5′-CCTTCA GCC CCG GGT ACT-3′ CEBP/α forw 5′-GAC CCT CAGCCTTGT TTGTAC TGT ATG CC-3′, CEBP/α rev 5′- TTT GGAAAG CTT GTC ATA ACT CCG GTC CC-3′ CEBP/β forw 5′- CCG CCCGTG GTG TTA TTT AAA GAA GAAA C GTC-3′, CEBP/β rev 5′- GCC CGTAGG AAC ATC TTT AAG CGA TTA CTC AG-3′ CEBP/δ forw 5′- CCA TCGACT TCA GCGCCT ACA TCG ACT C- 3′, CEBP/δ rev 5′-CCC GCCTTG TGA TTG CTG TTG AAG AGG T-3′ Glut-4 forw 5′-TTC CAACAG ATA GGC TCC GAA G-3′, Glut-4 rev 5′-AAG CAC CGC AGA GAA CAC AG-3′ HPRT forw 5′-GAG ATG GGA GGC CAT CAC ATT GTA GCC CTC-3′, HPRT rev 5′-CTC CAC CAA TTA CTT TTA TGT CCC CTG TTG ACT GGT C-3′ PPARγ forw 5′-GAT CCA GTG GTT GCA GAT TAC AA-3′, PPARγ rev 5′-GAG GGA GTT GGA AGG CTC TTC-3′ SREBP1C forw 5′-TTT CTGACA CGC TTC TTC CTG AGC AGT G-3′, SREBP1C rev 5′-ATG TTCCCG GAATAG CTG AGT CAC CTG G-3′.

Adipose tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and preincubated with quenching buffer (3% H₂O₂ in methyl alcohol) and blocking buffer (10% normal goat serum in PBS) for 30 min at room temperature. The samples were then treated with the primary antibodies against adiponectin (1:100, Thermo Scientific, IL, USA), PDGFRα (1:50, R&D System, MN, USA), UCP1 (1:300, Abcam, Cambridge, UK) and BrdU (1:100, Abcam) and with secondary Alexa Fluor 488- or 594-conjugated antibodies (Invitrogen). To detect BrdU staining, paraffin sections were incubated in 50% formamide 2× SSC (Welgene Inc., Daegu, Korea) for 2 h at 65 °C, washed with 2× SSC, and treated with 2 N HCl for 30 min at 37 °C. After washing with PBS, tissue sections were blocked with 10% normal goat serum (Vector Laboratories CA, USA). For nuclear staining, the samples were mounted on cover slips in DAPI-containing mounting medium (ImmunoBioScience, Burlingame, WA, USA). Images were captured using a confocal microscope system (LSM 710, Carl Zeiss, Oberkochen, Germany).