Membrane fraction proteins of Hep G2 and SK-Hep–1 cells were identified by MALDI-TOF.

<p>ND: None detected.</p><p><b>The functional classification (Class)</b> of identified proteins is shown a ~ m. a: cell cycle; b: chaperone/ stress response; c: cytoskeleton/ cell mobility; d: DNA replication/ gene regulation/ cell proliferation; e: ion channels; f: membrane proteins; g: metabolic enzyme; h: protection and detoxification; i: protein synthesis and degradation; j: signal transduction; k: transport/ binding proteins; l: intermediate filaments; m: unannotated/ function inferred.</p><p>Membrane fraction proteins of Hep G2 and SK-Hep–1 cells were identified by MALDI-TOF.</p

List of unregulated proteins in the 2DE map of either the well-differentiated HepG2 cells or the poorly differentiated SK-Hep–1 cells identified by MALDI-TOF MS.

<p>List of unregulated proteins in the 2DE map of either the well-differentiated HepG2 cells or the poorly differentiated SK-Hep–1 cells identified by MALDI-TOF MS.</p

Summary of the differentially expressed proteins in HepG2 and SK-Hep–1 cells using MALDI-TOF MS and categorized according to their functional roles.

<p><b>Note:</b> “+” sign denotes positive expression; “-” denotes negative expression</p><p>Summary of the differentially expressed proteins in HepG2 and SK-Hep–1 cells using MALDI-TOF MS and categorized according to their functional roles.</p

FLJ25439, a novel cytokinesis-associated protein, induces tetraploidization and maintains chromosomal stability via enhancing expression of endoplasmic reticulum stress chaperones

<p>Investigation of the mechanisms leading to aneuploidy and polyploidy is critical to cancer research. Previous studies have provided strong evidence of the importance of tetraploidization as an early step in tumorigenesis. In cancer cells, tetraploid cells may contribute to abnormal mitotic progression, which may be associated with cytokinesis failure. Tetraploidy leads to genomic instability due to centrosome and chromosome over-replication. Until now, the mechanism by which cells maintain tetraploid status has been unknown. Here, we identified a novel D box-containing protein, FLJ25439, which displays a dynamic expression profile during mitosis/cytokinesis with the midbody as the most prominent associated structure. To understand the function of FLJ25439, we established stable cell lines overexpressing FLJ25439. FLJ25439-overexpression cells grew slower and displayed a tetraploid DNA content in comparison with diploid parental cells. They also showed aberrant mitosis and dysregulated expression of p53, pRb and p21, suggesting a defect in cell cycle progression. To explore the molecular mechanisms responsible for FLJ25439-induced tetraploidization, we conducted a comparative analysis of the global protein expression patterns of wild type and overexpressors using proteomics and bioinformatics approaches. Protein category profiling indicated that FLJ25439 is involved in pathways related to anti-apoptosis, protein folding, the cell cycle, and cytoskeleton regulation. Specifically, genotoxic-stress- and ER stress-related chaperone proteins greatly contributed to the FLJ25439 overexpression phenotypes. The results of this study pave the way to our further understanding of the role of this novel cytokinesis-related protein in protecting cells from environmental stress and tetraploid formation.</p

Representative 2D gel images of various cellular compartments depicting identified protein spots that were differentially expressed between HepG2 and SK-Hep–1 clonal variants.

<p>(A) Total cell lysate, (B) cytosol, (C) nucleus, and (D) membrane. Differentially expressed proteins are numbered and boxed. Reference proteins are indicated by arrowheads.</p

Protein classification.

<p>The functional classifications of the identified proteins.a: Cell cycle: 3%; b: chaperone/stress response: 9%; c: cytoskeleton/ cell mobility: 15%; d: DNA replication/gene regulation/cell proliferation: 12%; e: ion channels: 2%; f: membrane proteins: 4%; g: metabolic enzyme: 16%; h: protection and detoxification: 6%; i: protein synthesis and degradation: 8%; j: signal transduction: 8%; k: transport/binding proteins: 6%; l: intermediate filaments: 15%; and m: unannotated/ function inferred: 4%.</p

The pathologic characteristics and expression levels of HSP 27 and ANX1.

<p>To understand the correlation between pathologic characteristics and expression levels of HSP 27 and ANX1, the immunohistochemical stains with HSP 27 and ANX1 in HCC tissue microarray were performed. HCC was divided into well-differentiated (WD), moderately differentiated (MD) and poorly differentiated (PD). (A) HSP27 expression levels were scored semiquantitatively as weakly positive (1+), moderately positive (2+) and strongly positive (3+). (B) Expression levels of ANX1 were scored semiquantitatively as negative, weakly positive and strongly positive. The association between pathologic characteristics and ANX1 expression was analyzed.</p

Validation of the differential protein expression between HepG2 and SK-Hep–1 cells by western blot.

<p>Actin was used as the internal control in the cytosolic fraction (A), PCNA served as the internal control in the nuclear fraction (B), and Na-K+ ATPase was used as the internal control in the membrane fractions (C). (D) C: cytosolic fraction protein, N: nuclear fraction protein, M: membrane fraction protein.</p

Cytosolic fraction proteins of Hep G2 and SK-Hep–1 cells were identified by MALDI-TOF.

<p>ND: None detected.</p><p><b>The functional classification (Class)</b> of identified proteins is shown a ~ m. a: cell cycle; b: chaperone/ stress response; c: cytoskeleton/ cell mobility; d: DNA replication/ gene regulation/ cell proliferation; e: ion channels; f: membrane proteins; g: metabolic enzyme; h: protection and detoxification; i: protein synthesis and degradation; j: signal transduction; k: transport/ binding proteins; l: intermediate filaments; m: unannotated/ function inferred.</p><p>Cytosolic fraction proteins of Hep G2 and SK-Hep–1 cells were identified by MALDI-TOF.</p

Nuclear fraction proteins of Hep G2 and SK-Hep–1 cells were identified by MALDI-TOF.

<p>ND: None detected.</p><p><b>The functional classification (Class)</b> of identified proteins is shown a ~ m. a: cell cycle; b: chaperone/ stress response; c: cytoskeleton/ cell mobility; d: DNA replication/ gene regulation/ cell proliferation; e: ion channels; f: membrane proteins; g: metabolic enzyme; h: protection and detoxification; i: protein synthesis and degradation; j: signal transduction; k: transport/ binding proteins; l: intermediate filaments; m: unannotated/ function inferred.</p><p>Nuclear fraction proteins of Hep G2 and SK-Hep–1 cells were identified by MALDI-TOF.</p