DF1 Cell Culture, Transfection, and Glycogen Detection

The chicken fibroblast DF1 cells were obtained from the cell bank of the Chinese Academy of Sciences. The DF1 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, United States) and 1% penicillin-streptomycin (Gibco). The cell lines were cultured at 37°C in a humidified incubator with 5% CO₂ (Li et al., 2020).

The FOSL2 (Gene ID: 421416) overexpressing lentivirus and empty vectors were constructed by Ubigene (Guangzhou, China) and denoted as YOE-LV001-FOSL2 and YOE-LV001-Ctrl, respectively. Cells were plated in six-well plates at a density of 1 × 10⁶ cells per well, and YOE-LV001-FOSL2 and YOE-LV001-Ctrl were transfected into DF1 cells by the Polybrene, a transfection-promoting reagent provided by Ubigene. The formula for virus dosage is V (μL) = 1,000 × MOI × N/T, where MOI is multiplicity of infection, N is number of cells, and T is lentiviral infectious titer.

The transfected cells were selected with 2 μg/mL puromycin for 2 days. Using an inverted microscope, the imaging system is Olympus BX41, and the images are taken at a 100× field of view. Then cells were collected and tested for glycogen content. The amount of glycogen in DF1 cells was quantified using a glycogen content assay kit (Solarbio, BC0340, China) according to the manufacturer’s instructions (Zhao et al., 2017). The cell experiment was repeated three times with samples in triplicate.