CHUNG, SOONKYU, Ph. D. Mechanisms by Which Conjugated ...

For real time qPCR, 1 ug total RNA was converted into first strand cDNA using
Omniscript RT kit (Qiagen Inc). qPCR was performed in a Smartcycler (Cepheid,
Sunnyvale, CA) using the QuantiTect SYBR Green PCR kit (Qiagen) for 40 cycles. To
account for possible variation related to cDNA input amounts or the presence of PCR
inhibitors, the endogenous reference gene glycerol aldehyde-3-phosphate dehydrogenase
(GAPDH) was simultaneously quantified in a separate tube for each sample. Initial real-
time amplifications were examined by agarose gel electrophoresis to confirm the sizes of
the products. After PCR amplification, a melting curve was generated for every PCR
product to check the specificity of the PCR. Primer sequences and running conditions are
summarized in Table 1.
Transient Transfection and PPAR Activity
For measuring PPARγ activity, primary human adipocytes were transiently
transfected with a 3X PPAR responsive luciferase reporter construct pTK-PPRE3x-Luc
(Kliewer et al. 1992; generously provided by Dr. Susanne Mandrup) using the Amaxa
Nucleofactor (Amaxa, Cologne, Germany) according to the manufacturer’s protocol. On
day 6 of differentiation, 1 x 10 6 cells from a 60 mm plate were trypsinized and
resuspended in 100 µl of nucleofector solution (Amaxa) and mixed with 2 µg of pTK-
PPRE3x-luc and 25 ng pRL-CMV for each sample. Electroporation was performed using
the V-33 nucleofector program (Amaxa). Cells were replated in 96-well plates after 10
minutes recovery in calcium-free RPMI medium. Two hours later, cultures were
supplemented with charcoal-stripped AM1 before LPS stimulation. Firefly luciferase
99