2.6. Generation of HOXB5-Expressing Cells

The coding sequence of HOXB5 was subcloned into the retroviral vector pDon-5 neo (Takara, Shiga, Japan), thus generating the HOXB5 overexpression vector. The coding sequence of HOXB5 and pDon-5 Neo were transfected into LNCaP cell lines by lipofection using Fugene-6 (Promega, Madison, WI, USA). After transfection, cells were released into drug-free medium for 48 h followed by G418 selection until single colonies were formed. Single clones were expanded, and overexpression of HOXB5 was confirmed by Western blotting.