Figure 7.

DUB-IN-2 hinders the deubiquitinating activity of USP48 and induces the degradation of HMGA2. (A) Relative protein level of HMGA2 in the screening of DUBs inhibitors. The SW480 cells were treated with the library of 45 reported DUB inhibitors at a concentration of 2.5 μmol/L for 12 h as indicated, and then, the protein levels of HMGA2 were assessed by Western blotting. (B) Schematic diagram of USP48 activity detection after treatment with compounds in vitro. (C) Ubiquitinated HMGA2 was immunoprecipitated from HEK293T cells transfected with Myc-HMGA2 and His-Ub plasmids with after 10 μmol/L MG132 treatment. Purified USP48 from HEK293T cells overexpressing HA-USP48 was pretreated with DUB inhibitors for 1 h and subsequently incubated with ubiquitinated HMGA2 for 6 h. Then, the ubiquitylation level of HMGA2 was evaluated. (D) HEK293T cells were co-transfected with MYC-HMGA2 and His-ubiquitin, and then treated with 2.5 μmol/L DUB-IN-2 24 h. After pretreatment with MG132 (10 μmol/L) for 6 h, anti-Myc beads were used for IP, and anti-Myc and His antibodies were used for IB analysis. (E) The deubiquitinating effect of USP48 on HMGA2 upon treatment with different concentrations of DUB-IN-2. HA-USP48 was exposed to 5, 2.5 and 1 μmol/L DUB-IN-2 and then incubated with ubiquitinated HMGA2. The ubiquitylation level of HMGA2 was measured. (F) IB analysis of HMGA2 and β-Tubulin in HCT116 and SW480 cells pretreated with 2.5 μmol/L DUB-IN-2 24 h and then treated with MG132 (10 μmol/L) for 6 h. (G, H) DLD1 and HEK293T cells were co-transfected with MYC-HMGA2, HA-USP48 and His-ubiquitin, and then treated with 2.5 μmol/L DUB-IN-2 24 h. After pretreatment with MG132 (10 μmol/L) for 6 h, anti-Myc beads were used for IP, and anti-Myc, anti-HA and His antibodies were used for IB analysis. (I) Purified HA-USP48 was pretreated with Spautin-1 (2.5 μmol/L) or DUB-IN-2 (2.5 μmol/L) for 10 min and then incubated with K48-linked Di-ubiquitin in the presence of the compound at 37 °C for 1.5 h. Samples were then analyzed by IB analysis with a ubiquitin-specific antibody. (J) IB analysis of HMGA2 and β-Tubulin in control and USP48-knockdown SW480 cells treated with 2.5 μmol/L DUB-IN-2 24 h. (K) Representative images of lung sections were stained with HE, and metastatic nodules were calculated in each group. Four-week-old BALB/c nude mice were injected intravenously with 2 × 10⁶ DLD1 cells with high USP48 expression. DUB-IN-2 (1 mg/kg) or vehicle was injected intraperitoneally on Day 8 and every other day for 5 weeks. Scale bar, 1 cm ∗∗∗P < 0.001.