ShRNA oligos of KPNB1 are purchased from Integrated DNA Technologies, Inc. (Coralville, IA,USA). Annealed double strand oligos were cloned into pSUPER.retro.puro vector (OligoEngine, Seattle, WA, USA) according to the provided manual for the expression of shRNA. The reconstructed plasmids were amplified in E.coli (New England Biolabs, Ipswich, MA, USA) and purified using plasmid miniprep kit (Thermo fisher scientific).
Virus was produced using Gryphon retroviral packaging cell lines (Allele Biotechnology, San Diego, CA, USA). In brief, 10 μg of plasmid was transfected to Grypho cell lines in a 6-cm dish. In 48 hours, supernatant that contains virus particles was collected. For virus infection, 1 ml of the collected virus supernatant supplemented with 3 μl of polybrene (EMD Millipore, Billerica, MA, USA) at 5 mg/ml was added to the culture medium of C42B in a 6-cm plate. In 24 hours, virus supernatant was replaced with fresh medium. Infected cells were selected using puromycin (Sigma-Aldrich-Aldrich, St. Louis, MO, USA).
Virus was produced using Gryphon retroviral packaging cell lines (Allele Biotechnology, San Diego, CA, USA). In brief, 10 μg of plasmid was transfected to Grypho cell lines in a 6-cm dish. In 48 hours, supernatant that contains virus particles was collected. For virus infection, 1 ml of the collected virus supernatant supplemented with 3 μl of polybrene (EMD Millipore, Billerica, MA, USA) at 5 mg/ml was added to the culture medium of C42B in a 6-cm plate. In 24 hours, virus supernatant was replaced with fresh medium. Infected cells were selected using puromycin (Sigma-Aldrich-Aldrich, St. Louis, MO, USA).