Plasmid Construction for AMPK Studies

An expression plasmid for CA-AMPK^{53 (link)} was kindly provided by Dr. Rusty Jones (McGill University) and the insert was transferred to pBabe-puro and pWZL-blast. Expression plasmids for mouse C/EBPβ coding region (C/EBPβ^ΔUTR) and C/EBPβ coding region plus 3′UTR (C/EBPβ^UTR) have been described previously^{10 (link)}; the genes were transferred to pBabe-puro and pWZL-blast retroviral vectors. Lentiviral expression vectors for AMPKα2 and CaMKKβ were provided by Dr. Dominic Esposito (FNLCR, Frederick, MD). Lentiviral vectors containing an shRNA construct simultaneously targeting AMPKα1/2 (human and mouse)^{54 (link)} and a control shRNA vector were kindly provided by Dr. Thomas Brown (Wright State University). Lentiviral shRNA vectors targeting mouse CaMKKβ (#TRCN0000276649), AMPKα1 (#TRCN0000360770) and Importin α1 (#TRCN0000093514) were purchased from Sigma-Aldrich. Human C/EBPβ shRNA and a non-targeting shRNA control in pSuperRetro-neo were described previously^{10 (link)}. Human HRAS^G12V was expressed from pWZL-hygro^{6 (link)}. Plasmids for retroviral packaging have been described^{9 (link)}; lentiviral packaging/envelope plasmids pMD2.G (#12259), pMDLg/pRRE (#12251), and pRSV/Rev (#12253) were obtained from Addgene.

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Basu S.K., Gonit M., Salotti J., Chen J., Bhat A., Gorospe M., Viollet B., Claffey K.P, & Johnson P.F. (2018). A RAS-CaMKKβ-AMPKα2 pathway promotes senescence by licensing post-translational activation of C/EBPβ through a novel 3′UTR mechanism. Oncogene, 37(26), 3528-3548.

Publication 2018

Lentiviral shRNA vectors Importin α1 pMD2.G pMDLg/pRRE pRSV/Rev

Genes Human Importin Mouse Plasmids Retroviral Shrna Vectors

Corresponding Organization :

Other organizations : National Cancer Institute, Center for Cancer Research, Frederick National Laboratory for Cancer Research, Institute on Aging, National Institute on Aging, National Institutes of Health, Institut Cochin, Inserm, UConn Health

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Variable analysis

independent variables

CA-AMPK
Mouse C/EBPβ coding region (C/EBPβ^ΔUTR^)
Mouse C/EBPβ coding region plus 3'UTR (C/EBPβ^UTR^)
AMPKα2
CaMKKβ
ShRNA construct simultaneously targeting AMPKα1/2 (human and mouse)
ShRNA targeting mouse CaMKKβ
ShRNA targeting mouse AMPKα1
ShRNA targeting mouse Importin α1
Human C/EBPβ shRNA
Human HRAS^G12V^

dependent variables

Not explicitly mentioned

control variables

Control shRNA vector

controls

Positive control: None specified
Negative control: Control shRNA vector

Annotations

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