Shrna targeting

MHT cells were cultured in DMEM supplemented with 10% fetal bovine serum, streptomycin (0.1 mg/ml), and penicillin (100 U/ml) as previously described [35 (link)]. Malat1 and control ASOs, and the ROCK inhibitor Y27632 (Tocris, Cat. No. 1254) were added to cells at the indicated concentrations for 24 hours. 5 nmole of Silencer select siRNA (Ambion, Negative Control #1, Lpin1 –s66131, ADI1 –s75892 and 186988, VAMP3 –s98387 and s98386) was formulated in RNAiMAX plus Opti-MEM (ThermoFisher Scientific) and incubated with cells for 48 hrs. shRNA targeting Vamp3 was purchased from Origene (Cat. Nos. scrambled control: TR30021V, Vamp3_1: TL515358VC, Vamp3_2: TL515358VD). MHT cells cultured as described to a 50% confluency were exposed to 20uL viral particles for 24hrs. Cells were washed and selected with 1ug/mL Puromycin (Sigma Aldritch, Cat. No. P8833) for 10 days, then colonies were selected and plated in a 96 well plate. Target knockdown was assessed by QPCR and normalized to cyclophilin.

Full-length of NTKL was cloned into PCDNA3.1 and stably transfected into QGY-7703 (NTKL-7703) and MHC-97H cells (NTKL-97H) using lipofectamine2000 (Origene, Rockville, MD). Empty vector-transfected cells (Vec-7703 and Vec-97H) were used as controls. NTKL depleted cells were established by stably transfecting shRNA targeting NTKL (Origene, Rockville, MD) into QGY-7703 cells.