Lentiviral Vector Production and Transduction

All plasmids were purified utilizing a GenElute HP Plasmid Midiprep Kit (Sigma) after transformation using DH5α cells with ampicillin. Lentiviral packing plasmids, pMD2.G and psPAX2, were purchased from Sigma. LentiCRISPRv2 was obtained as a gift from Dr. Anil Rustgi (Columbia University, New York, NY, USA). The single-guide RNA (sgRNA) sequence targeting the MEN1 gene (Supplemental Table S1) was cloned into the lentiCRISPRv2 vector as previously described [6 (link)]. shRNA plasmids for LXRα were obtained from the University of Pennsylvania Perelman School of Medicine High-Throughput Screening Core (Philadelphia, PA, USA), all of which were derived from a pLKO.1-puromycin backbone, with sequences for the LXRα shRNAs listed in Supplemental Table S1. The pLKO-Tet-On was obtained from Addgene and used to generate doxycycline-inducible menin shRNAs using two validated menin shRNA sequences (Supplemental Table S1) as previously described [23 (link),24 (link)]. To produce lentivirus, 293T cells were transfected with pMD2.G, psPAX2, and the plasmid of interest using Fugene 6 (Promega, Madison, WI, USA) according to the manufacturer’s instructions. After collecting and filtering the virus, cells were then transduced in the presence of 4 μg/mL polybrene (hexadimethrine bromide). Twenty-four hours after completion of transduction, cells were then selected with puromycin for 72 h.