Plasmid-Mediated Autophagy Regulation in Prostate Cancer

Viral packaging plasmids (pRSV-REV, pVSV-G, pMDL-g/p-RRE) were obtained from AddGene. ATG5 short hairpin RNA (shRNA) constructs (TRCN0000151963, TRCN0000151474, TRCN0000330392), ATG7 shRNA constructs (TRCN0000007586, TRCN0000007588, TRCN0000364479), Sigma1 shRNA constructs (TRCN0000296908, TRCN0000291305, TRCN0000061010, TRCN0000061008), and a nontargeting control shRNA construct (SCH002) were obtained from Sigma-Aldrich. The FLAG-AR and FLAG-ARV7 plasmid constructs were gifts from Dr. Stephen Plymate (University of Washington School of Medicine, Seattle, WA) and have been described elsewhere (58 (link)). pEGFP-LC3 was a gift from Drs. Grazia Ambrosini and Gary K. Schwartz [Memorial Sloan Kettering Cancer Center (MSKCC), New York, NY] and has been described previously (56 (link)). Transient transfections were performed with jetPRIME transfection reagent (PolyPlus) according to manufacturer's procedures. LNCaP GFP-LC3 stable cell lines were generated using the pBABE-Puro-mCherry-EGFP-LC3B plasmid construct (Addgene 22418). Transfection was performed with jetOPTIMUS transfection reagent (PolyPlus) according to manufacturer's protocol. Cells were maintained in high-glucose RPMI (Corning) supplemented with 10% FBS (Corning) and 1 µg/mL puromycin (Thermo Fisher Scientific).

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Oyer H.M., Steck A.R., Longen C.G., Venkat S., Bayrak K., Munger E.B., Fu D., Castagnino P.A., Sanders C.M., Tancler N.A., Mai M.T., Myers J.P., Schiewer M.J., Chen N., Mostaghel E.A, & Kim F.J. (2023). Sigma1 Regulates Lipid Droplet–Mediated Redox Homeostasis Required for Prostate Cancer Proliferation. Cancer Research Communications, 3(10), 2195-2210.

Publication 2023

pRSV-REV pVSV-G pMDL-g/p-RRE SCH002 jetPRIME transfection reagent jetOPTIMUS transfection reagent FBS puromycin

Cancer Cell lines Cells Gifts Glucose Plasmid Puromycin Shrna Transfection Transient

Corresponding Organization :

Other organizations : Thomas Jefferson University, Sidney Kimmel Cancer Center, Janssen (United States), University of Washington, VA Puget Sound Health Care System, Geriatric Research Education and Clinical Center, United States Department of Veterans Affairs

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Variable analysis

independent variables

ATG5 short hairpin RNA (shRNA) constructs (TRCN0000151963, TRCN0000151474, TRCN0000330392)
ATG7 shRNA constructs (TRCN0000007586, TRCN0000007588, TRCN0000364479)
Sigma1 shRNA constructs (TRCN0000296908, TRCN0000291305, TRCN0000061010, TRCN0000061008)
FLAG-AR and FLAG-ARV7 plasmid constructs
PEGFP-LC3 plasmid construct

dependent variables

Outcomes not explicitly mentioned

control variables

Viral packaging plasmids (pRSV-REV, pVSV-G, pMDL-g/p-RRE) from AddGene
A nontargeting control shRNA construct (SCH002) from Sigma-Aldrich
LNCaP GFP-LC3 stable cell lines generated using the pBABE-Puro-mCherry-EGFP-LC3B plasmid construct (Addgene 22418)
Cells maintained in high-glucose RPMI (Corning) supplemented with 10% FBS (Corning) and 1 µg/mL puromycin (Thermo Fisher Scientific)

positive controls

None specified

negative controls

A nontargeting control shRNA construct (SCH002) from Sigma-Aldrich

Annotations

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