Animal experiments. Male and female CD1 mice (8 weeks of age) were purchased from Charles River Laboratories International Inc. (Wilmington, MA), housed in microisolator cages in a temperature-controlled room (22–24°C) with a 12-hr light, 12-hr dark cycle, and provided water and food (standard low-fat diet for rodents RMH 2500; Purina Mills, Richmond, IN) ad libitum. All animals were treated humanely with regard for alleviation of suffering, and all procedures were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. For prenatal chronic chemical exposure, dimethylsulfoxide (DMSO) (0.1%), rosiglitazone (ROSI) (0.5 µM), or TFZ (0.1, 1.0, or 10.0 µM) were supplemented in the drinking water during breeding and throughout pregnancy. Carboxylmethyl cellulose (CMC) at 0.5% was added to the water to increase the solubility of test chemicals, and control animals received water containing 0.5% CMC and DMSO vehicle. After the pups were born, normal filtered tap water was provided. The pups were kept together with their dams after birth and weaned at 3 weeks of age. Experimental mice were weighed and sacrificed at 8 weeks of age, and adipose tissues (epididymal fat for male and ovarian fat for female, retroperitoneal fat, and subcutaneous fat) were collected, weighed, and used for MSC production.
The vectors pCMX-GAL4 and pCMX-GAL4-mPPARγ were previously described (Grun et al. 2006
). Transient transfections were performed in COS7 cells as described by Chamorro-Garcia et al. (2012)
. Briefly, COS7 cells were seeded at 15,000 cells/well in 96-well tissue culture plates in 10% calf bovine serum. The following day, cells were transfected in Opti-MEM reduced-serum medium (all media and reagents from Invitrogen Life Technologies, Grand Island, NY unless noted otherwise) at approximately 90% confluency. One microgram of CMX-GAL4 effector plasmid was co-transfected with 5 µg tk-(MH100)4
-luciferase reporter and 5 µg CMX-β-galactosidase transfection control plasmids using Lipofectamine 2000 reagent, following the manufacturer’s recommended protocol. After overnight incubation, the medium was replaced with Dulbecco’s modified Eagle medium (DMEM)/10% resin charcoal–stripped fetal bovine serum (FBS) (Tabb et al. 2004
) plus ligands at concentrations indicated in the figure legends for an additional 24 hr before luciferase and β-galactosidase assays (Milnes et al. 2008
). All transfections were performed in triplicate and reproduced in multiple experiments. Data are reported as fold induction over vehicle (0.1% DMSO) controls (mean ± SE) for triplicate samples (three biological replicates) and results were verified in multiple experiments.
3T3-L1 cells were maintained in DMEM supplemented with 10% FBS, 2 mM l
-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin and differentiated as described previously (Li et al. 2011
) using various concentrations of DMSO, ROSI, and TFZ. Briefly, cells were cultured until 2 days postconfluence, at which time the adipogenic induction cocktail MDI (IBMX, dexamethasone, and insulin) plus test ligands was added (Li et al. 2011
). After 2 days, the medium was replaced with fresh medium containing test ligands and incubation continued for 5 additional days. For antagonist experiments, 1 µM T0070907 (Cayman Chemical, Ann Harbor, MI) was supplemented into the media every 12 hr. At the end of the experiment, cells were fixed and stained with Oil Red O to visualize lipid accumulation, or collected for RNA extraction, followed by QPCR [quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR)] for gene expression analysis as described previously (Li et al. 2011
). For spontaneous differentiation assays, 3T3-L1 cells were incubated at 2 days postconfluence in culture media supplemented with the indicated chemicals for 7 days.
Human white adipose tissue–derived MSCs were purchased from Lonza (Basel, Switzerland), cultured, and differentiated as described by Kirchner et al. (2010)
. Briefly, postconfluent cultures were treated with adipogenic or osteogenic induction cocktails together with test ligands or vehicle controls. Antagonist treatment was as noted above. Fourteen days (adipogenic) or 21 days (osteogenic) after differentiation was initiated, cells were stained with Oil Red O or collected for RNA extraction.
Mouse adipose tissue–derived MSCs were collected from the epididymal fat pads of male mice and cultured as described by Kirchner et al. (2010)
Total RNA was isolated from cells and tissues using TRIzol reagent as recommended by the manufacturer (Invitrogen Life Technologies). Reverse transcription and QPCR were performed using Transcriptor reverse transcriptase and Sybr Green Master Mix (Roche Diagnostics Corp., Indianapolis, IN) (Li et al. 2011
). The following genes were examined: ADIPOQ
(fatty acid binding protein 4), FSP27
(fat-specific protein of 27 kDa), LEP
(leptin), and LPL
(lipoprotein lipase). [For details of the sequences of primers used for QPCR, see Supplemental Material, Table S1
Statistical analysis. Data are presented as mean ± SE. One-way ananlysis of variance (ANOVA) was used to determine the difference of means in relative mRNA abundance, staining, body weights, or adipose depot weights among TFZ treatment groups and negative control (DMSO). This was followed by a Dunnett’s post hoc test to ascertain statistical significance for each TFZ-treatment group compared with control (DMSO). The unpaired t-test was used to determine the significance of effects elicited by the positive control, ROSI relative to DMSO. Additional statistics were calculated for some experiments: One-way ANOVA with Bonferroni post hoc test was conducted comparing +T0070907 versus –T0070907 with each other. A p-value of < 0.05 was considered statistically significant. Statistical analysis used GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA).