Adenoviral Delivery of GFP-LC3B for Autophagy Monitoring

cDNA for LC3B was obtained by reverse transcription-polymerase chain reaction (RT-PCR). Briefly, total RNA was isolated from keratinocytes using an easy-BLUE RNA extraction kit (Intron, Daejeon, Korea), after which 2 μg of total RNA was reverse-transcribed with the Moloney-murine leukemia virus (M-MLV) reverse transcriptase (Elpis Biotech, Daejeon, Korea). An aliquot of RT mixture was then subjected to PCR amplification using a primer set for LC3B (5′-GTACGGATCCATGCCGTCGGAGAAGACCTT and 5′-AATTCTCGAGTTACACTGACAATTTCATCC). The amplified full-length cDNA for LC3B was then subcloned into the pENT/cytomegalovirus-green fluorescent protein (GFP), and replication-incompetent adenoviruses were created as reported previously [16 (link)]. For monitoring autophagy, the cells were transduced with adenovirus expressing GFP-LC3B overnight, after which the cells were replenished with fresh growth medium and incubated for an additional 24 h. After treatment with poly (I:C), autophagy was monitored by observation of LC3 puncta under the fluorescent microscopy.