Figure 2. PVT1 in exosomes inhibits degradation and ubiquitination of ERG in osteosarcoma cells. Ssos-2 and MNNG/HOS cells were transfected with siRNA of PVT1 (si-PVT1) for 48 h. (A) The expression of ERG protein. (B) The degradation of ERG protein at 3, 6, and 9 hours after the treatment of the protein synthesis inhibitor, CHX (125 μg/mL). (C) The SPOP and ERG proteins were detected in PVT1-protein complex using RNA pull-down assay. Input was used as the positive control; antisense RNA was used as the negative control. (D) PVT1 was detected in ERG-RNA binding complex using RIP assay. Input was used as the positive control; IgG was used as the negative control. (E) Ubiquitination assay: Ssos-2, MG-63, and MNNG/HOS cells were transfected with pcDNA-PVT1, HA-Ub and His-SPOP for 24 h followed by the immunoprecipitation with HA antibody and immunoblotting with ERG antibody. (F) The ubiquitination assay was also performed in PVT1-interfering osteosarcoma cells after being co-cultured with BMSC-EXO. Three independent experiments. *p<0.05, **p<0.01 vs si-control. CHX, cycloheximide. pcDNA-PVT1, the PVT1 overexpressing vector. HA, hemagglutinin. Ub, ubiquitin.