FIGURE 7 from Gigaxonin Suppresses Epithelial-to-Mesenchymal Transition of Human Cancer Through Downregulation of Snail

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Gigaxonin-mediated regulation of Snail in gene transfected ME180 cells. A, FLAG-Snail and Myc-GAN plasmid DNA cotransfected ME180 cells show higher expression of FLAG and Snail in cells split 48 hours after transfection pointing to the expression of both exogenous (FLAG-Snail) and endogenous Snail in transfected cells. Expression of higher molecular weight GAN protein (140 kD) is reduced in these cells possibly containing higher fraction of fast growing cells. The unsplit cells show higher expression of gigaxonin (both lower and higher molecular weight proteins) due to the presence of slow growing cells in the population. These cells however have lost expression of FLAG-Snail and Snail confirming giagxonin mediated downregulation of exogenous and endogenous Snail. Reduced actin expression indicates that gigaxonin could be involved in actin ubiquitination. B, Immunoprecipitation with gigaxonin and hybridization to Snail shows hybridizing band to be same size as that of IgG immunoprecipitated proteins. Thus, we believe that there is no direct interaction between gigaxonin and Snail. C, Treatment of ME180 and GAN edited cells with inhibitors of proteosome (10 µmol/L each of MG132 and NEM) and autophagy (100 nmol/L of bafilomycin A) pathways shows increased expression of Snail in proteosome inhibitor–treated GAN edited cells in comparison to that of DMSO control treated cells pointing to the involvement of gigaxonin in Snail downregulation. As a confirmation of proteosomal inhibitor activity, there is increased expression of cyclin D1 (both ME180 and GAN edited cells) and NFκB of ME180/C4.15.1 cells in MG132/NEM-treated cell lines. Reduced expression of cyclin D1, p16, and gigaxonin after bafilomycin A treatment indicates blockage of cells, as expected at G1–S boundary, by bafilomycin A. The above results suggest that gigaxonin downregulates Snail through an indirect pathway, likely through NFκB. Numbers at the bottom of the protein bands indicate ratio of band intensity versus β-actin. Although there is reduced β-actin in proteosomal inhibitor lane (fifth lane), there is higher protein band intensity in both Snail and NFκB in comparison with DMSO-treated cells. This is also reflected in band intensity/β-actin numbers. D, Immunofluorescence analysis of ME180 cells shows endogenous expression of GAN and Snail in the cytoplasm, surrounding the nucleus. GAN nuclear expression was seen in 10% to 20% of cells (indicated by arrows). While overlap of the two proteins is seen in many cells, there are also cells expressing Snail or GAN alone pointing to the expression of both proteins in ME180 cells.

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