PVT1 binds to the FOXM1 protein and increases its protein level by enhancing its stability. PVT1 binds to the FOXM1 protein and increases its protein level.

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PVT1 binds to the FOXM1 protein and increases its protein level by enhancing its stability. PVT1 binds to the FOXM1 protein and increases its protein level by enhancing its stability. A, SDS-PAGE with Coomassie brilliant blue staining (left) showed the proteins pulled down by PVT1 or its antisense RNA from H293T cells. The arrow indicates the possible FOXM1 in the PVT1-bound complex, the peptides of which were detected by subsequent LS/MS mass spectrometry. The FOXM1 protein was detected by Western blotting using the corresponding antibody in another independent RNA pull-down assay (right). B, RIP assays showed that FOXM1 interacted with PVT1 in H293T and HGC27 cells. Fifteen percent of the cell lysates as input and NOP2 were used as positive controls, and IgG was used as a negative control. The RT-qPCR products were analyzed with RNA electrophoresis (left). The mRNA levels were graphed (right). *, P < 0.01. C, Western blot results suggested that the protein level of FOXM1 was elevated in PVT1-overexpressing AGS and MGC803 cells but reduced in PVT1-knockdown HGC27 and MKN45 cells. D, Representative images of the immunohistochemical (IHC) analysis of PVT1-knockdown MKN45 and HGC27 cells. E, RT-qPCR results showed that overexpression and knockdown of PVT1 failed to affect the mRNA level of FOXM1 in gastric cancer cells. F, AGS cells were transfected with the indicated plasmids for 48 hours. Cycloheximide (CHX; 50 μg/mL) was then added at 0, 1, 3, and 5 hours. Lysates were collected at the indicated time points and immunoblotted with the indicated antibodies. The intensities of Western blot results that reflected the remaining protein levels of FOXM1 were quantified by ImageJ software and graphed. The experiments were performed in triplicates (right: *, P < 0.01). G, HGC27 cells were transfected with siNC or siPVT1 for 48 hours. MG132 (10 μmol/L) was added for 3 hours before harvesting. Lysates were immunoblotted with the indicated antibodies. Mi-die Xu et al. Clin Cancer Res 2017;23:2071-2080 ©2017 by American Association for Cancer Research