ROC curves show that SPG20 is a biomarker for colorectal carcinomas and adenomas. The area under the ROC curve (AUC) signifies the accuracy of the SPG20 biomarker for distinguishing colorectal carcinomas (blue line) and adenomas (green line) from normal colorectal tissue samples. AUC, area under the curve; ROC, receiver operating characteristics; SPG20, spastic paraplegia-20.

The SPG20-encoded protein Spartin is not expressed in hypermethylated colon cancer cell lines. (a) Western blot analysis showing levels of Spartin expression in colon epithelial cell lines. (b–g) Immunofluorescence confocal images of endogenous Spartin (red) in the indicated cell lines. The pictures in panels b and d–g are generated using identical microscopy settings for comparison of the Spartin level. The arrow points to a cytokinesis bridge (enlarged in panel c). The inset in panel c shows an overlay with phase-contrast image to visualize the midbody. Nuclei are shown in blue. The cells are fixed with 3% PFA. Size bars: 20 μm. PFA, paraformaldehyde; SPG20, spastic paraplegia-20.

Colon cancer cell lines show several cytokinesis profiles. (a) The relative numbers of cells were quantified from immunofluorescence pictures using the Olympus Scan R microscope. (b) The cells were stained for α-tubulin and Aurora-B to visualize cytokinesis profiles. The immunofluorescence pictures were used to quantify the percentage of cells in cytokinesis. The error bars show the ±s.e.m. of four independent experiments. In total, more than 3000 cells were counted for the CRL-1790 control line, approximately 10 000 for HT29, and SW480 and between 30 000 and 40 000 for the HCT116 and RKO lines. (c–f) Three-dimensional reconstructions of immunofluorescence confocal z-stack images showing the difference in the amount of cytokinesis profiles between the four colon cancer cell lines (c, RKO; d, HCT116; e, SW480; f, HT29). Aurora-B (red), α-tubulin (green) and nuclei (blue). The arrow points to a cytokinesis profile. The cells were fixed with 3% PFA. PFA, paraformaldehyde.

Rescue of Spartin expression results in the normalization of the cytokinesis process. Identical number of CRL-1790 cells (a–d) and HT29 cells (f–i) were seeded out on coverslips and treated or not with 1 μ AZA for 3 days as indicated in the figure. The cells were fixed with 3% PFA and stained for DNA (blue) and endogenous Spartin (red). (c, d, h, i) Magnified views of the areas indicated by white squares. (e, j) Western blots showing endogenous Spartin in AZA-treated versus that in non-treated cells. HT29 cells were treated (l) or not (k) with AZA, fixed with 3% PFA and stained for Aurora-B (red), α-tubulin (green) and DNA (blue). The difference in the number of cytokinesis profiles between AZA-treated and non-treated cells (arrowheads k, l) is shown in panel m. (n) Quantitations of relative growth rates. The error bars show the ±s.d. of four independent coverslips. For each condition, approximately 3000 cells were quantified for CRL-1790 and 30 000 cells for HT29. AZA, 5-aza-2′deoxycytidine; PFA, paraformaldehyde.

Promoter methylation of SPG20 is associated with loss of gene expression. (a) Gene expression and DNA promoter methylation status of SPG20 in cell lines. The quantitative gene expression levels are shown as ratios between the median of SPG20 and the average of two endogenous controls, GUSB and ACTB. (b) Relative gene expression of SPG20 in colon cancer cell lines treated with 1 μ AZA for 72 h, 0.5 μ TSA for 12 h and a combination of both drugs (AZA+TSA). AZA, 5-aza-2′deoxycytidine; SPG20, spastic paraplegia-20; TSA, trichostatin-A.

Spartin localizes to the spindle poles and the cytokinesis bridge of dividing cells. Three-dimensional reconstructions of immunofluorescence confocal images showing the localization of endogenous Spartin (red) to the cytokinesis bridge of HeLa cells (a–f) and to the spindle poles of prometaphase in HeLa (g, h, k) and CRL-1790 (i, j) cells. α-Tubulin (a–c, g–i) or γ-tubulin (j, k) is shown in green. Nuclei are shown in blue. Yellow indicates colocalization. The cells were pre-permeabilized with 0.05% saponin prior to fixation with 3% PFA (a–i) or fixed directly in methanol (j, k). Size bars: 5 μm. PFA, paraformaldehyde.

‘Slower' growing colon cancer cell lines have convoluted cytokinesis bridges. A three-dimensional reconstruction of immunofluorescence confocal z-stack images showing the morphology of cytokinesis profiles in SW480 cells (a–c) and HT29 cells (d–e). Aurora B (red), α-tubulin (green) and nuclei (blue). In addition to having convoluted cytokinesis bridges, both cell lines show distribution of Aurora-B along the bridge, indicative of late cytokinesis profiles. The cells were pre-permeabilized with 0.05% saponin prior to PFA fixation (a, b) or fixed directly with 3% PFA. Size bars, 5 μm. PFA, paraformaldehyde.