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Fig. 2 | Journal of Hematology & Oncology

Fig. 2

From: Overexpression of CLC-3 is regulated by XRCC5 and is a poor prognostic biomarker for gastric cancer

Fig. 2

XRCC5 was identified as a CLC-3 promoter-binding protein, and both CLC-3 and XRCC5 were prognostic factors of the overall survival in GC patients. a The basic RNA expression of CLC-3 in cell lines (n = 3). b Truncated fragments of the CLC-3 gene promoter were designed and amplified by PCR to construct dual-fluorescence reporter plasmids. c The promoter activity of various reporter plasmids was measured by dual-luciferase reporter assay (n = 3). d A 5′-biotin-labeled DNA probe with a promoter region between − 248 and + 226 was synthesized. e Potential CLC-3 promoter-binding proteins in nuclear protein extracts were pulled down using the synthesized probe. After SDS-PAGE and silver staining, the target protein band was observed (indicated with the red rectangle), which was significantly enriched in GC cells and finally analyzed by mass spectrometry. f Binding between XRCC5 and the CLC-3 promoter was detected by WB in the nuclear protein/DNA complex using a synthesized probe or nonspecific probe (NSP). g Binding of XRCC5 to the CLC-3 DNA was confirmed by ChIP assay. The PCR products were separated on 2% agarose gels. h Representative images of XRCC5 and CLC-3 expression in GC tissues and ANT. i The expression of XRCC5 in 90 GC tissues was higher than that in ANT. j The expression of XRCC5 and CLC-3 was positively correlated in GC tissues. k High expression of XRCC5 predicted a poor prognosis for GC patients. l There was no difference in overall survival between GC patients with high CLC-3 and low XRCC5 levels and GC patients with high XRCC5 and low CLC-3 levels. m GC patients with high expression of XRCC5 and CLC-3 had the worst prognosis. *P < 0.05, **P < 0.01

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