4.6. RNA Interference

The lentiviral vectors pLKO.1-shLacZ (TRCN0000072223: 5′-TGTTCGCATTATCCGAACCAT-3′) and pLKO.1-shMAP1B (#1, TRCN0000116621: 5′-GCCTGGAATAAACAGCATGTT-3′; #2, TRCN0000290688: 5′- CCCTGACTTAGGAGTTGTATT-3′) were obtained from the Taiwan National RNAi Core Facility (Taipei, Taiwan) and used to establish stable MAP1B-silenced clones of RTCC1 and J82 cell lines using shRNAs against MAP1B (shMAP1B).Viruses were produced by transfecting HEK293 cells with the above three vectors using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) [29]. For viral infection, 3 × 10⁶ RTCC1 and J82 cells were incubated with 8 mL of lentivirus in the presence of polybrene, followed by puromycin selection of the stable clones of lentivirus-transduced cells.