Pmd2 g

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.

HEK293T cells were grown in 10‐cm dishes to 80% confluency before transfection with the lentiviral vector (10 μg) with packaging vectors including pMD2.G (3 μg, Addgene plasmid # 12259), psPAX2 (6 μg, Addgene plasmid # 12260) and pAdVAntage (3 μg, E1711, Promega) using Lipofectamine 2000 Transfection Reagent (11668019, Thermo Fisher Scientific) according to the manufacturer's protocol. After 16 h, medium was refreshed. Supernatant containing lentivirus was harvested at 24 and 48 h after medium refreshing and pooled together. Supernatant was centrifuged at 300 g to remove cell fragments and passed through 0.45 μm filter. Lentivirus containing > 10 kb length insert (inducible CRISPRi) was concentrated using AVANTI J‐30I centrifuge (Beckman Coulter) with JS‐24.38 swing rotor at 72,000 g for 2 h at 4°C and pellets were dissolved in 200 μl PBS. Other lentivirus, including individual gRNAs, constructively active β‐catenin and SOX9 inducible overexpression, were concentrated using Lenti‐X™ Concentrator (631232, Takara) and pellets were dissolved in 400 μl PBS.