Misson shrna

PARP-1 (MISSON shRNA, Sigma) or control shRNA (pLKO.1) lentivirus vector were co-transfected with the VSV-G envelope and psPAX2 packaging plasmids (Addgene) into human embryonic kidney (HEK) 293T cells using Lipofectamine 2000 (Thermo Scientific). After 48 h, lentivirus-containing supernatants were harvested and centrifuged at 1000 × g for 5 min to discard the debris. THP-1 cells were infected with the virus for 24 h and then selected by 1 µg/ml puromycin for 72 h. Knockdown efficacy was analyzed by immunoblotting.

The sequences of shRNAs described below were obtained from the database of MISSON® shRNA (Sigma-Aldrich). shRNAs against NIPSNAP1 (5′-GATCCAGTTTCACAATGTAAA-3′) and NIPSNAP2 (5′-GTGTTGCCAAAGATTCACGAA-3′) were cloned into Tet-PLKO-puro (Addgene; 21915) according to Addgene’s protocol. The Tet-PLKO-puro plasmid was used as a control. Supernatants containing shRNA-encoding lentiviruses were collected, and the copy number was quantified using a qPCR Lentivirus Titer Kit (Applied Biological Materials Inc., British Columbia, Canada). Then, lentiviruses were transduced into BEAS-2B cells at a multiplicity of infection of 5. shRNA-integrated cells were selected by culture in maintenance medium (LHC9 medium containing 0.5 μg/mL puromycin, 100 U/mL penicillin, and 100 μg/mL streptomycin) for 1 week. Then, cells were maintained using maintenance medium or cultured in medium supplemented with 100 ng/mL DOX (FUJIFILM Wako) for 2 weeks, during which time the medium was changed every 2 days.