Lentiviral shRNA Knockdown

The virus was produced from Phoenix Ampho cells using Mirus Bio TransIT-2020 and cotransfected with various short hairpin RNA (shRNA) expression vectors in combination with pMD2.G and psPAX2 vectors and the pLKO.1-shRNA expression vectors. The short interfering RNA sequences targeting LacZ, CEBPD, and AMPK were subcloned into the lentiviral expression vector pLKO.1. The short interfering RNA sequences are as follows: shLacZ (shZ): 5′-CCGGTGTTCGCATTATCCGAACCATCTCGAGATGGTTCGGATAATGCGAACATTTTTG-3′; shCEBPD (shD): 5′-CCGGGCCGACCTCTTCAACAGCAATCTCGAGATTGCTGTTGAAGAGGTCGGCTTTTT-3′; shAMPKα (shKα1): 5′-CCGGTGATTGATGATGAAGCCTTAACTCGAGTTAAGGCTTCATCATCAATCATTTTT-3′; shAMPKα (shKα2): 5′-CCGGCAACTTTACCTGGTTGATAACCTCGAGGTTATCAACCAGGTAAAGTTGTTTT-3′. The expression vectors and shRNAs were obtained from the National RNAi Core Facility located at the Genomic Research Center of Institute of Molecular Biology, Academia Sinica, Taiwan.