Sh usp7

The small interference RNA (siRNA) targeting MTDH or USP7 (si-MTDH or si-USP7) or the short hairpin RNA (shRNA) targeting USP7 (sh-USP7) were designed by GenePharma with nontargeted siRNAs or shRNAs as the negative control (si-NC or sh-NC). MTDH-overexpression plasmids (MTDH) were established by cloning full-length of MTDH into pcDNA3.1 plasmids (GenePharma), and the empty pcDNA3.1 plasmids were used as the negative control (pcDNA). The transient transfection was performed using the Lipofectamine 3000 (Invitrogen).

All transfection experiments were performed as previously described [47 (link)]. Plasmids shSNAI2, shUSP7 and shTRIM21 and their lentiviral packaging were commissioned for Genepharma (Shanghai, China) synthesis, and overexpressed plasmids (FLAG-tagged SNAI2, FLAG- or HA- or Myc-tagged USP7/wt, Myc-tagged USP7/C223S, HA-Ub/mt, HA-Ub/wt, Myc-tagged TRIM21) and their lentiviral packaging were commissioned for OBiO (Shanghai, China). siUSP7 and negative controls were purchased from GenePharma. RD and A204 cells were infected with leniviruses, and stable cells were screened with 2ug puromycin. The shRNA sequences used were as follows: shNC:5’-GTTCTCCGAACGTGTCACGT-3’;shSNAI2:5’-CCAGAATGTCTCTCCTGC-3’;shUSP7:5’-CCTGGATTTGTGGTTACGTTA-3’;shTRIM21:5’-TGAGAAGTTGGAAGTGGAAAT-3’; siNC: 5’-UUCUCCGAACGUGUCACGU-3’; USP7 siRNA-1:5’-ACCCUUGGACAAUAUUCCUTT-3’;USP7 siRNA-2: 5’-AGUCGUUCAGUCGUCGUAU-3’.