Overexpression of ESRP2 in HepG2 and AML12 cells

HepG2 and AML12 cell lines were obtained from ATCC (Catalogue no. HB-8065 and CRL-2254, respectively) and cultured according to specifications as stated by ATCC. When tested for mycoplasma contamination (Biotool, catalogue no. B39032) prior to use, these cell lines tested negative. cDNAs encoding FLAG-tagged mouse Esrp2^{43 (link)} were sub-cloned into the p-AdenoX-ZsGreen1 vector (Clonetech, 632267) using the InFusion kit (Clonetech, 639646) as per the manufacturer’s instructions. High-titer adenoviruses were generated by transfecting Ad-293 cells (~ 70% confluent) in T-25 flasks with linearized recombinant adenoviral plasmid using Mirus TransIT-2020 kit and cells were harvested once cytopathic effect (CPE) was seen. Following this, two viral amplification steps were performed and the viral particles were purified using CsCl gradient as mentioned in the Adeno-X^™ Adenoviral System 3 user manual. After purification of viral particles, the titer was determined by UV spectrophotometry at 260 nm. To determine the effect of overexpression of ESRP2 in HepG2 and AML12 cells, T-25 flasks containing ~50% confluent HepG2 and AML12 cells were infected with 1.5 X 10⁹ opu (optical particle units) of the ESRP2 or GFP adenovirus for 48 hours and cells were harvested to extract RNA and protein for further analysis.