Figure 5.

SKP2 mediated the invasion of BC cells by promoting the degradation of the Sp1 protein. (A) Real-time PCR was applied to compare the Sp1 mRNA levels in T24T(Vector) and T24T(KOSOX2 C1/C2) cells. (B,E). The indicated cells were pretreated with MG132 and the cells were then subjected to protein degradation assay in the presence of CHX for the indicated time points. Western blot was used to analyze Sp1 protein degradation rates between the indicated cells. β-Actin or GAPDH was used as a protein loading control. (C) The indicated stable transfectants were subjected to Western blot to determine SKP1, SKP2, HSP90, HSP 70, and ITCH protein levels. (D) The indicated stable transfectants were subjected to Western blot to determine Sp1, FOXO1, and HUR protein levels. (F,G) The invasion abilities of T24T(KOSOX2/Vector) and T24T(KOSOX2/myc-SKP2) cells were determined using a BD BioCoat^TM Matrigel^TM Invasion Chamber. The asterisk (*) indicates a significant difference in invasion abilities in comparison to its vector control transfectant (p < 0.05). The bars are presented as the mean ± SD from three independent experiments. (H) Real-time PCR was applied to compare the mRNA levels of SKP2 in T24T(Vector) and T24T(KOSOX2 C1/C2) cells. The asterisk (**) indicates a significant difference in SKP2 mRNA in comparison to its vector control transfectant (p < 0.01). (I) The indicated cells were transfected with SKP2 promoter-driven luciferase reporter together with pRL-TK. The transfectants were seeded into 96-well plates and then subjected to determine SKP2 promoter luciferase activity. pRL-TK was used as an internal control to normalize transfection efficiency. Each bar indicates the mean ± SD from three replicate assays. (J) The diagram shows the transcription factors that could potentially bind to the human HUR promoter region. (K) The 293T cells with overexpressing SOX2 were tested by ChIP assay using an anti-SOX2 antibody to test the interaction of SOX2 with SKP2 mRNA.