Establishing Stable Cell Lines for NOV Expression/Knockdown in 3T3-L1 Adipocytes

3T3-L1 mouse pre-adipocytes were purchased from the ATCC (Manassas, VA, USA). After thawing, 3T3-L1 cells were cultured as previously described [26 (link),35 (link)]. For studies aimed at achieving adipocyte cell-specific overexpression or knockdown of NOV, 1 × 10⁶ cells were seeded in six-well plates. Adipocyte (3T3-L1) cells were transfected for 3 h with 1 × 10⁶ transducing units (TF) of overexpressing NOV (ORF-NOV) or NOV knockdown (shNOV) lentivirus (Vector builder, Shenandoah, TX, USA) to establish a stably transduced cell line. Cells were also treated with the transduction medium without lentiviral particles, which served as the un-transduced control. After 48 h of incubation, the antibiotic selection medium (α-MEM growth medium with 10 µg/mL puromycin) was used to select for transduced cells. Cells were then cultured and maintained as described above [26 (link),32 (link),33 (link)].