Metafectene easy

Mouse embryonic fibroblasts [21 (link)] were trypsinized after reaching 70% confluence and transfected using Metafectene Easy (Biontex Laboratories, Germany) according to the manufacturer's recommendations. For each well (in a 96-well plate) a total of 0.05 µg of DNA were used (0.015 µg pM-hPPARγ-LBD + 0.03 µg Gal4-responsive luciferase reporter + 0.0025 µg pRL-CMV normalization vector). Dulbecco's modified Eagle's medium (DMEM), media containing vehicle (0.1% DMSO), positive control (1 μM Rosi), or extract dissolved in DMSO was added to the cells after 6 h of transfection. Cells were washed with phosphate buffered saline (PBS) after 18 h of transfection and lysed with lysis buffer (0.2 M KH₂PO₄, 0.2 M K₂HPO₄, 0.4% Triton N-101, 100 µM phenylacetic acid, and 100 µM oxalic acid). Photinus and Renilla luciferase activities were measured directly in the plate as previously described [20 (link)]. Photinus activities were normalized to corresponding Renilla values to compensate for differences in transfection efficiency. Results are presented as the mean ± SD.

We used the mammalian expression plasmid vector pSilencer 4.1-CMV puro (Life Technologies) to produce a hairpin siRNA molecule for OCT4. The target sequence of siRNA for OCT4 was 5′-AGGAGCACGAGTGGAAAGCAA-3′. The construct was made by ligating complementary oligonucleotides that create the stem–loop structure of 21 bp into the BamHI and HindIII sites of the pSilencer 4.1-CMV vector. The construction was confirmed by DNA sequencing. The pSilencer 4.1-CMV control plasmid containing a scrambled siRNA was used as a negative control (scr siRNA, Life Technologies). Cells were transfected with the different constructs using Metafectene Easy+ (Biontex), according to the manufacturer’s instructions. Twenty-four hours post transfection, transfected cells were selected using puromycin (2.5 µg/mL).