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Supplementary Figure 1 from SPINDLIN1 Promotes Cancer Cell Proliferation through Activation of WNT/TCF-4 Signaling

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posted on 2023-04-03, 17:44 authored by Jing-Xue Wang, Quan Zeng, Lin Chen, Ji-Chao Du, Xin-Long Yan, Hong-Feng Yuan, Chao Zhai, Jun-Nian Zhou, Ya-Li Jia, Wen Yue, Xue-Tao Pei

PDF file - 2MB, Effect of SPINDLIN1 depletion on cancer cell proliferation and WNT/TCF-4 signaling. A. The siRNA interference sequence against SPINDLIN1 or an oligonucleotide control sequence was transfected into HeLa cells, and RT-PCR was used to examine the expression of SPINDLIN1, using �-ACTIN as an internal standard. B. HeLa cells treated with the same methods as A were used for the CCK-8 assay to analyze cell proliferation. The absorbance at 450 nm was monitored every 24 h over 7 d. N=6. C. Five hundred HeLa cells per well were treated with the same methods as A and were plated in 6-well plates and incubated for 2-3 weeks. Colonies were stained with crystal violet (top panels) and the numbers of colonies were counted (bottom panel). N=3, **p<0.01 compared to control (Student's t test). D. In HeLa cells, pTOPFlash was co-transfected with the siRNA interference sequence against SPINDLIN1 or the negative control sequence, and the pFOPFlash control vector was co-transfected with the siRNA interference sequence against SPINDLIN1. The TCF/�-CATENIN-responsive activity was expressed as relative luciferase activity, normalized to Renilla activity, and given as n-fold relative to the activity of control. Error bars indicate the means � SD. N=3, **p<0.01 compared with the level of OT+siRNA-SPINDLIN1 (Student's t test).

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ARTICLE ABSTRACT

SPINDLIN1, a new member of the SPIN/SSTY gene family, was first identified as a gene highly expressed in ovarian cancer cells. We have previously shown that it is involved in the process of spindle organization and chromosomal stability and plays a role in the development of cancer. Nevertheless, the mechanisms underlying its oncogenic role are still largely unknown. Here, we first showed that expression of SPINDLIN1 is upregulated in clinical tumors. Ectopic expression of SPINDLIN1 promoted cancer cell proliferation and activated WNT/T-cell factor (TCF)-4 signaling. The Ser84 and Ser99 amino acids within SPINDLIN1 were further identified as the key functional sites in WNT/TCF-4 signaling activation. Mutation of these two sites of SPINDLIN1 abolished its effects on promoting WNT/TCF-4 signaling and cancer cell proliferation. We further found that Aurora-A could interact with and phosphorylate SPINDLIN1 at its key functional sites, Ser84 and Ser99, suggesting that phosphorylation of SPINDLIN1 is involved in its oncogenic function. Collectively, these results suggest that SPINDLIN1, which may be a novel substrate of the Aurora-A kinase, promotes cancer cell growth through WNT/TCF-4 signaling activation. Mol Cancer Res; 10(3); 326–35. ©2012 AACR.

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