Molecular Cloning and Manipulation of Androgen Receptor

FOXA1 siRNA (sense 5′-GAGAGAAAAAAUCAACAGC-3′; antisense 5′-GCUGUUGAUUUUUUCUCUC-3′)^{14 (link),20 (link)}, AR on-target plus smart pool (L-003400-00-0020), and control siRNA Luciferase GL2 Duplex (D-001100-01-20) were synthesized by Dharmacon. The control and pGIPZ lentiviral shRNAmir targeting FOXA1 (Clone ID# V2LHS_16780) were purchased from Open Biosystems. C-terminal 3×HA-tagged full-length FOXA1 was cloned into a Tet-On inducible lentiviral vector that was kindly provided by Professor Junjie Chen (M.D. Anderson Cancer Center). The full-length (wild-type) and truncated AR (Q640X) were amplified from pcDNA3.1-AR^fl plasmid^{24 (link)} and cloned into the pCR8/GW/TOPO entry vector (Invitrogen). The pCR8-AR C562S and G568W mutants were generated from pCR8-AR wildtype using QuikChange® II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). Lentiviral constructs were generated by LR recombination between pCR8-AR constructs and pLenti CMV/TO Puro DEST (Addgene plasmid 17293). Details regarding cloning primers and plasmid construction were provided in Supplementary Table 1 and Supplementary Table 2.