2.13. Cell culture and transient transfections

NIH3T3 cells (American Type Culture Collection) were cultured in DMEM (Mediatech), containing 10% fetal bovine serum (Gemini Bio), and 1x penicillin-streptomycin (Life Technologies/Invitrogen) in a humidified 5% CO₂ incubator at 37 °C. For luciferase assays, NIH3T3 cells were seeded into 24-well plates (Nunc) at 30,000 cells per well. Transient transfections were performed using PolyJet™ (SignaGen Laboratories, Rockville, MD), following manufacturer's recommendations. NIH3T3 cells were co-transfected as indicated in the figure legend with 200 ng/well luciferase reporter plasmids, as well as 100 ng/well thymidine kinase-β-galactosidase reporter plasmid, which served as an internal control [44]. Six total Bmal1-luciferase plasmids were used. The full-length mouse Bmal1-luciferase reporter plasmid (Addgene, Plasmid #46824) was modified to remove an erroneous homeodomain binding site at −537 bp which does not appear in either the mouse genome or in the original depositor's sequence. This modified Bmal1-luciferase plasmid was used for all experiments. Site directed mutagenesis of the ATTA-like sites in the Bmal1-luciferase plasmid was performed using the NEB Q5 Site-Directed Mutagenesis Protocol (New England Biolabs Inc.), following manufacturer's instructions. Primers for mutagenesis were designed using NEBase Changer (Supplementary Table 2). The expression vector used was mouse Six3/pcDNA overexpression plasmid (200 ng/well, Origene Technologies, Rockville, MD). To equalize the amounts of DNA transfected into cells, we systematically equalized plasmid concentrations by adding the corresponding plasmid backbone. Cells were harvested 24 h after transfection in lysis buffer [100 mM potassium phosphate (pH 7.8) and 0.2% Triton X-100]. Luciferase values were normalized to β-galactosidase values to control for transfection efficiency. Values were further normalized by expression as fold change compared to pcDNA (the empty expression vector control plasmid), as indicated in the figure legend. Data represent mean ± SEM of at least five independent experiments done in triplicate.