Journal: Journal of Virology

Article Title: Digitoxin Suppresses Human Cytomegalovirus Replication via Na + , K + /ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

doi: 10.1128/JVI.01861-17

Figure Lengend Snippet: Digitoxin fails to inhibit HCMV in ATG5 knockdown (KD) cells. (A) ATG5-deficient cells were generated using shRNA, and KD efficiency was confirmed by WB. (B) pLKO.1 control cells and ATG5 KD cells were infected and treated with digitoxin (30 nM), and bafilomycin A1 (50 nM) was added 4 h before harvest. The expression of LC3-II, p62, and ATG5 was detected by WB. (C) Control pLKO.1 and ATG5 KD cells were infected with HCMV TB40 at 200 PFU/well and treated with digitoxin or GCV in a 12-well plate. Plaques were counted after 10 days, and data are presented as means ±SD.

Article Snippet: The following sequences were used to generate Na + ,K + /ATPase α1 subunit short hairpin RNA (shRNA) and a nonsilencing (NS) control by cloning into the lentiviral vector pLKO.1 hygro (Addgene): clone 1, 5′-GCCTTTCAGAACGCCTATTG-3′; clone 2, 5′-ATACCTTAACCAGTAACATTC-3′, and NS, 5′-CCTAAGGTTAAGTCGCCCTCG-3′.

Techniques: Generated, shRNA, Infection, Expressing

Journal: Journal of Virology

Article Title: Digitoxin Suppresses Human Cytomegalovirus Replication via Na + , K + /ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

doi: 10.1128/JVI.01861-17

Figure Lengend Snippet: Digitoxin fails to induce autophagy or to inhibit HCMV in α1 KD cells. (A) Control and α1 KD cells were infected and treated with digitoxin (30 nM), AICAR (0.8 μM), digitoxin plus AICAR, or GCV. Levels of pp65, pAMPK, and p62 were detected by WB at 72 hpi. (B) Uninfected cells were treated with digitoxin or GCV. Levels of pAMPK and p62 were detected by WB. (C) α1 KD and control HFFs were infected with HCMV Towne (100 plaques/well), followed by treatment with digitoxin or GCV. Plaques were counted under each condition at day 8 postinfection. (D) (Left) Lysates from panel A were used to detect mTOR, p-mTOR, and the mTOR substrates p-Ser S6K and p-Thr S6K. (Right) p-mTOR levels were quantitated by densitometry. (E) pLKO.1 and α1 KD cells were infected and treated with digitoxin or GCV for 12 h. One-milligram aliquots of protein from the prepared lysates were used to pull down ULK1. AMPK, mTOR, and α1 were detected by WB of the input samples.

Article Snippet: The following sequences were used to generate Na + ,K + /ATPase α1 subunit short hairpin RNA (shRNA) and a nonsilencing (NS) control by cloning into the lentiviral vector pLKO.1 hygro (Addgene): clone 1, 5′-GCCTTTCAGAACGCCTATTG-3′; clone 2, 5′-ATACCTTAACCAGTAACATTC-3′, and NS, 5′-CCTAAGGTTAAGTCGCCCTCG-3′.

Techniques: Infection