Figure 6.

The function of USP48 on HMGA2 is significantly enhanced by SUMOylation at Lys258. (A) IB analysis of total lysates and anti-HA (left panel) or anti-Myc immunoprecipitates (right panel) from DLD1 cells transfected with control vector or Flag-SENP5. (B) IB analysis of total lysates and anti-HA immunoprecipitates derived from DLD1 cells transfected with the control vector, Myc-HDAC4, or Myc-HDAC7, as indicated. (C) IB analysis of total lysates and anti-Myc immunoprecipitates derived from HEK293T cells transfected with HA-USP48 WT or K258R and Myc-HMGA2, as indicated. (D–E) Representative images are shown with merged PLA and DAPI channels from PLA experiments. Scale bar, 10 μm. Each red dot represents the detection of the USP48–HMGA2 interaction complex, and the graphs representing mean ± SD are shown in (E). (F–G) HEK293T cells were transfected with HA-USP48 WT or K258R. After fixation, in situ PLA for USP48-HMGA2 was performed with anti-HA and anti-Myc antibodies. Scale bar, 10 μm. Each red dot represents the detection of the USP48-HMGA2 interaction complex, and the graphs representing mean ± SD are shown in (G). (H) IB analysis of total lysates and anti-Myc immunoprecipitates of HEK293T cells transfected with His-Ub, HA-USP48 WT/K258R and Myc-HMGA2 and treated with MG132 (10 μmol/L for 6 h), as indicated. (I–J) IB analysis of total lysates and anti-Myc immunoprecipitates of HEK293T cells transfected with Myc-HMGA2, His-Ub, HA-USP48, and control vector, Flag-SENP5 (I), Myc-HDAC4 (J), or Myc-HDAC7 (J) and treated with MG132 (10 μmol/L for 6 h), as indicated. (K) DLD1 cells were transfected with specific plasmids, as indicated and treated with CHX (50 μg/mL) for the indicated time points. Quantification of HMGA2 levels relative to β-Tubulin is shown. (L) DLD1 cells stably expressing USP48 shRNA were transfected with indicated plasmids and treated with 200 μmol/L 2-D08 or DMSO for 24 h. Graphic representation of the migration ability from cells described above was examined by transwell migration assay. Scale bar, 200 μm. (M) 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 cells as in (I). 2-D08 (4 mg/kg) or vehicle was injected intraperitoneally on Day 8 and every other day for 5 weeks. Scale bar, 1 cm. Data are presented as mean ± SD. One-way ANOVA test (E). Unpaired two-tailed Student's t-test (G). All experiments were performed independently at least three times. ∗∗∗P < 0.001, ns indicates no statistical significance.