Maxiprep

Transient transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in 1.0 to 1.5 × 10⁶ cells/well plated onto 24-well tissue culture plates. For each fluc reporter construct used in transfections, two independent plasmid Maxipreps (Qiagen, Valencia, CA) were used. Each transfection consisted of 0.8 μg DNA of fluc reporter construct and 0.4 μg SV40-Renilla luciferase (Rluc) vector, bearing Rluc under the SV40 promoter (Promega, Madison, WI), as an internal control of transfection efficiency. Luciferase activity was analyzed 24 hrs post-transfection, using a dual luciferase reporter kit (Promega) and a well-plate luminometer. Luciferase assays were repeated at least four times, and within each experiment each construct was transfected in quadruplicate. Transfection of mammalian constructs bearing the coding region of the GCH1 (WTGCH1, SNPGCH1 and 141GSNPGCH1) was performed in 0.5 to 1 × 10⁶ cells/well plated onto 6-well tissue culture plates, using 5 μg of each DNA per well. Twenty-four hours post-transfection, cells from each transfection were spitted, one fraction was processed for immunoblotting and the rest was re-plated onto coverslips, allowed to attach and fixed for immunocytochemistry.

An Ig κ-chain signal sequence (METDTLLLWVLLLWVPGSTGDG), AP tag (GGLNDIFEAQKIEWHEGATG) and SEP were cloned in-frame with the 5'-end of the coding sequence for the mature rat GluA1 and 2 subunit proteins. The entire open reading frames (ORFs) were cloned upstream (5’) of an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence. The BirA-ER coding sequence30 (link) (a gift from A. Ting, MIT Cambridge) was then cloned to the 3' end of the IRES such that the start codon of the BirA-ER signal sequence corresponded to the 11th ATG of the IRES sequence. Doxycycline-dependent expression of the resulting dual-construct bAP::SEP::GluA was achieved by cloning the entire AP::SEP::GluA IRES BirA-ER sequence into the multiple cloning site of the pBI vector (BD Bioscience) and co-transfecting it with approximately equal molar quantities of rtTA-transactivator. The GluA2 subunit used was edited at the Q/R site (R607), unedited at the R/G site (R764) and the ligand-binding domain splice variant was flop except for residue S775 (flip), where amino acid numbering corresponds to the coding sequence of the immature GluA2 peptide (NP_001077280.1). The GluA1 subunit used was flip splice variant. Plasmid DNA was prepared using endotoxin-free MaxiPreps (Qiagen). All constructs were verified by restriction enzyme digest patterns and Sanger DNA sequencing.

A pre-designed shRNA vector set was purchased from Biosettia with shRNAs designed against SIRT3 (Gene ID 23410; Accession NM_001017524.2) in the pLV-H1-CMV-Green plasmid (Biosettia #SORT-B01). NEB stable competent E. Coli cells (New England BioLabs #C3040) were transformed by heat shock with plasmids obtained from Biosettia. Transformed colonies were picked and expanded under antibiotic selection. Plasmids were prepared from bacterial cultures by Maxiprep (QIAGEN #12165). OptiMEM (GIBCO) and Lipofectamine 2000 (Invitrogen #11668019) were used to transfect 9 μg of pLV-H1-CMV-Green plasmid, 6 μg psPAX2 plasmid (Addgene #12260), and 3 μg pMD2.G plasmid (Addgene #12259) into LentiX 293T packaging cells in antibiotic free medium. pMD2.G and psPAX2 plasmids were generous gifts from Didier Trono. Lentivirus containing media was then collected, filtered and used with polybrene to infect target MDA-MB-231 Parental cells. Transduced cells were selected for using fluorescence associated cell sorting (FACS) to generate stable shRNA cell lines.