Figure 2.

USP48 maintains HMGA2 stability. (A) Increasing amounts of HA tagged USP48 were transfected into HEK293T cells along with equal amounts of Myc-HMGA2. Cell lysates were analyzed using IB and antibodies against Myc. “+” denotes 2.5 μg plasmid transfection, while “++” indicates 5 μg. (B) Two independent shRNAs were employed to silence USP48 expression in SW480 and HCT116 cells, followed by IB analysis of HMGA2 protein levels. (C) USP48-WT or USP48–C98S was overexpressed in DLD1 cells, followed by analysis of HMGA2 protein levels. (D) Equal amounts of Myc-HMGA2 and either USP48-WT or USP48–C98S were co-transfected into HEK293T cells, followed by IB analysis using Myc antibodies. (E) DLD1 cells were transduced with HA-tagged USP48-WT alone or in combination with USP48 shRNA, and HMGA2 levels were analyzed by IB analysis. (F) HCT116 cells were transduced with USP48 shRNA and either HA-tagged USP48-WT or USP48–C98S, followed by IB analysis to analyze HMGA2 levels. (G) qRT-PCR analysis was conducted to assess the expression of HMGA2 mRNA in SW480 cells with depleted endogenous USP48 via two independent shRNA. (H) The expression of HMGA2 mRNA was assessed by qRT-PCR in DLD1 cells following transduction with either vector control or HA-USP48. (I) qRT-PCR was performed to evaluate the expression levels of Slug, IGF2BP2 and SOX2 in SW480 cells transduced with two distinct HMGA2 shRNA constructs. (J) The expression levels of Slug, IGF2BP2 and SOX2 were quantified by qRT-PCR in SW480 cells that had been transduced with USP48 shRNA. (K) SW480 cells transfected with two independent USP48 shRNA were treated with DMSO, MG132 (10 μmol/L), or CQ (20 mmol/L) for 6 h, followed by IB analysis of USP48 and HMGA2. (L) DLD1 cells transfected with vector control, or HA-USP48 were treated with DMSO or MG132 (10 μmol/L) for 6 h, and then USP48 and HMGA2 were analyzed. (M) DLD1 cells were transfected with vector control, HA-USP48 or USP48-C98S, treated with 50 μg/mL of CHX, collected at the indicated times, and then subjected to IB analysis with antibodies against USP48 and HMGA2. Quantification of HMGA2 levels relative to β-Tubulin is shown. (N) SW480 cells with stable expression of either control shRNA or USP48 shRNA were treated with 50 μg/mL CHX, harvested at indicated time points, and analyzed by IB using antibodies against HMGA2 and USP48. Quantification of HMGA2 levels relative to β-Tubulin is presented as mean ± SD. (O) HCT116 stably expressing control shRNA or USP48 shRNA were treated with 50 μg/mL CHX, harvested at the indicated times, and then subjected to IB with antibodies against HMGA2 and USP48. Quantification of HMGA2 levels relative to β-Tubulin is shown. One-way ANOVA test (G, I, J, M‒O). Unpaired two-tailed Student's t-test (H). All experiments were performed independently at least three times. ∗∗∗P < 0.001, ns indicates no statistical significance.