Fig 2: Effects of 14-3-3s on inhibition of Kaiso's target gene CDH1 expression. (A) The Pearson correlation coefficient between mRNA levels of 14-3-3 family members and Kaiso target genes in public RNA-seq datasets from Cancer Cell Line Encyclopaedia (CCLE, 1156 cancer cell lines) and Genotype-Tissue Expression (GTEx, 11 688 human normal tissues). (B) The level of CDH1 in SGC7901 and MKN45 cells with or without wildtype Kaiso and 14-3-3s overexpression. The experiment was performed three times. (C) The level of the CDH1 mRNA in SGC7901 and MKN45 cells with or without wildtype Kaiso and 14-3-3s overexpression (each experiment was repeated at least three times). Data were presented as the mean ± SD (n = 3). P-values in Student's t-test are labelled (**P < 0.01, ns: nonsense). (D) the amounts of pT606-Kaiso, total Kaiso, 14-3-3s and CDH1 proteins in gastric carcinoma (T) and the paired normal tissues (N) from 12 patients by western blotting. (E) Correlation between ratios of pT606-Kaiso to total Kaiso and CDH1 to GAPDH proteins based on the density of these proteins in western blot. Spearman's rho coexpression coefficient (r) and P-value are shown (n = 24).

Fig 3: Immunofluorescence analysis of 14-3-3 proteins in Snail1 and Mock cells.Representative confocal microscopy images showing the expression and localization of 14-3-3 ß, e and s (red) in SW480-ADH Mock and Snail1 cells. GFP expression (green) was unaltered by Snail1 and was used as a control. Scale bar, 10 µm.

Fig 4: Increased level of 14-3-3t protein in involved compared with uninvolved psoriatic skin. Protein extracts prepared from keratome biopsies taken from involved and uninvolved psoriatic skin were analysed with Western blot technique. (A) Shows the membrane probed with an antibody detecting 14-3-3t. ß-actin was added to demonstrate equal protein loading. Pt. 1–4 is randomly chosen between the nine patients totally included. (B) Shows the protein level of all seven 14-3-3 isoforms. Band intensity was quantified using Kodak 1D Image Analysis Software. The protein level was normalized to ß-actin and uninvolved biopsies were indexed to one. Bars indicate mean ± SD. *P<0.05.

Fig 5: Kaiso interacted with 14-3-3 family members and accumulated in cytoplasm. (A) After the plasmids of 14-3-3 family members and GST-Kaiso were co-transfected into MGC803 cells, GST-Kaiso pulled down various isoforms of 14-3-3 family, especially 14-3-3s. The experiment was performed three times. (B) Endogenous Kaiso immunoprecipitated several 14-3-3 family members in MGC803 cell in Co-IP analysis. (C) 14-3-3s protein immunoprecipitated endogenous Kaiso in MGC803 cell in Co-IP analysis. Co-IP assay for the interaction of Kaiso and 14-3-3s was performed three times. (D) The subcellular location of endogenous Kaiso in MGC803 cells with or without 14-3-3? or 14-3-3s overexpression by indirect immunofluorescence staining assay. As labelled, scale bar for IF = 5 µm. The experiment was performed three times. (E) Proportion of Kaiso in the nucleus and in cytoplasm of MGC803 cells with and without 14-3-3? or 14-3-3s overexpression. N = 2274/1129/476 in the group of Ctrl/14-3-3?/14-3-3s. P-values in chi-square tests are displayed. (F) Western blot for detecting the amounts of endogenous Kaiso in cytoplasm and nucleus protein in MGC803 cells with 14-3-3s overexpression. The experiment was performed three times. (G) Comparison of the levels of GFP-Kaiso (wild-type) or T606A mutant in the cytoplasm and nucleus in MGC803 cells with and without 14-3-3s overexpression. Red and Green arrows: makeable increased and decreased Kaiso in the cytoplasm and nucleus by 14-3-3s. The experiment was performed three times.