Lentiviruses containing shrnas

Lentiviruses containing shRNAs targeting human SMURF2 were obtained from GeneChem. Cells were seeded 12 h prior to infection, and to establish stable cell lines, cells were selected using puromycin (2 μg/mL) for at least two weeks. Gene overexpression was achieved by transfecting cells with the corresponding plasmids, and the cells were plated in six-well plates at 1 × 10⁶ cells/well. Three siRNAs targeting ID2 were designed and synthesized (Beijing Tsingke Biotech Co., Ltd.) with the following sequences:
siID2-1 (Sense): 5′-CGAUGAGCCUGCUAUACAA-3′,
siID2-1 (Anti-sense): 5′-UUGUAUAGCAGGCUCAUCG-3′;
siID2-2 (Sense): 5′-GACUGCUACUCCAAGCUCA-3′,
siID2-2 (Anti-sense): 5′-UGAGCUUGGAGUAGCAGUC-3′;
siID2-3 (Sense): 5′-CUUCUGAGUUAAUGUCAAA-3′,
siID2-3 (Anti-sense): 5′-UUUGACAUUAACUCAGAAG-3′.
All transfection procedures were performed using Lipofectamine 2000 Reagent (11668-019, Invitrogen), according to the manufacturer's instructions. Plasmids were mixed with the transfection reagent and then added collectively to the corresponding cell lines. After 6 h of transfection, the supernatant was replaced with fetal bovine serum (FBS)-containing medium and cells were cultured for 24 h. The establishment of all cell lines was verified using RT-qPCR and western blotting.

The full-length sequence of circ7379 was amplified and cloned into a circRNA-specific overexpression lentiviral vector, GV689 (GeneChem, Shanghai, China), which contained two homology arms upstream and downstream of the circRNA sequence to promote circRNA cyclization. DHX9, RUNX1 and KSRP overexpression plasmids (GV367) were also purchased from GeneChem (Shanghai, China). Three siRNAs targeting the BSJ sites of circ7379 were designed and synthesized by GenePharma (Suzhou, China). SiRNAs targeting DHX9, RUNX1, and KSRP were also purchased from GenePharma (Suzhou, China). Lentiviruses containing shRNAs targeting circ7379 and RUNX1 were purchased from GeneChem (Shanghai, China). Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used for the cell transfection according to the manufacturer's instructions. After the transfection, stable HCT116 and SW480 cells were selected with puromycin (3 μg/mL; Gibco, Grand Island, NY, USA) for one week, and the surviving cells were continuously cultured as stable cells. The siRNA and shRNA sequences are listed in Supplemental Table 2.

Lentiviruses containing shRNAs targeting Par3 and 14-3-3ζ were purchased from Shanghai GeneChem (GeneChem, China) and used to infect cells. The shPar3 target sequence was 5′-GCCATCGACAAATCTTATGAT-3′ and the sh14-3-3ζ target sequence was 5′-GCAATTACTGAGAGACAACTT-3′. Cells infected with a non-effective scrambled shRNA with vector were used as controls. Puromycin (2 μg/mL) was used to select stable clones. After treated with puromycin 2 weeks, multiple single colonies were used in the following experiments.