Shscr

Recombinant lentiviruses expressing shSET7/9, shTRIM21, shSCR, FLAG-SET7/9, FLAG-TRIM21, FLAG-RUNX2, or FLAG-Vector were obtained from Shanghai GenePharma and used according to the manufacturer’s instructions. The concentrated lentiviruses were used to infect cells with 8 μg/ml polybrene in a 60-mm dish (5 × 10⁵ cells per well). The cells were selected with 2 μg/ml puromycin (Merck) and subjected to sorting.

Small interfering RNA targeting METTL3 (si-METTL3), and negative control (NC) were purchased from RiboBo (Guangzhou, China). The METTL3 interfering sequence was 5′-GCTGCACTTCAGACGAATT-3′. Human METTL3, MYC over-expression plasmids (OE-METTL3, OE-MYC), and negative control vector (Ctrl) were purchased from GenePharma (Shanghai, China). The plasmid vectors expressing shRNA that targets human METTL3 (sh-METTL3) and the scrambled shRNA (sh-Scr) were purchased from GenePharma (Shanghai, China). The shRNA sequences were as follows: sh-METTL3: 5′-GCACTTGGATCTACGGAATCC-3′, sh-Scr: 5′-GCTTCGCGCCGTAGTCTTA-3′. Plasmids for expression of wild-type METTL3 (METTL3-WT) and methyltransferase catalytic mutant METTL3 (METTL3-Mut) were kindly provided by Dr. Jun-Ju He (Central South University, China). Transfections were performed using Lipofectamine 2000 (Invitrogen, USA) for small interfering RNA and Lipofectamine 3000 (Invitrogen, USA) for plasmid according to the manufacturer's protocols.

Transfection was performed using cells in the logarithmic growth phase. The cells are seeded into six-well plates at a density of 5 × 105 cells/well. After the cells grow to 60% of the area of each well, transfection is performed. The specific procedure was carried out with Lipofectamine 3,000 according to the manufacturer’s instructions (Invitrogen, Thermo Scientific, MA, United States). The shRNA targeting TTN-AS1 (shTTN-AS1) and the shRNA targeting scramble (shSCR) were obtained from Shanghai GenePharma Co., Ltd (Shanghai, China).