Clinicohistopathological implications of phosphoserine 9 glycogen synthase kinase-3β/ β-catenin in urinary bladder cancer patients

Figure 1 Flow chart of the number and selection of patients/controls in the study population.

Figure 2 Graphical representation of percent immunohistochemical expression of marker proteins in 90 tissues of low stage (non-muscle invasive bladder cancer)/high stage (muscle invasive bladder cancer) and low grade/high grade tumors. A: pS9GSK-3β [Normal expression: strong membranous expression in urothelium; Aberrant expression: low membranous, strong nuclear and cytoplasmic, no expression in urothelium]; B: β-Catenin [Normal expression: strong membranous expression in urothelium; Aberrant expression: low membranous, strong nuclear and cytoplasmic, no expression in urothelium]; C: β-Catenin + pS9GSK-3β; D: Cyclin D1 [Normal expression: strong nuclear expression in mesenchymal area; Aberrant expression: strong nuclear expression in mesenchymal area]; E: Cyclin D1 + pS9GSK-3β + β-Catenin; F: Snail [Normal expression: strong nuclear expression in mesenchymal area; Aberrant expression: strong nuclear expression in mesenchymal area]; G: Snail + pS9GSK-3β + β-Catenin; H: Slug [Normal expression: strong nuclear expression in mesenchymal area; Aberrant expression: strong nuclear expression in mesenchymal area]; I: Slug + pS9GSK-3β + β-Catenin. Numbers mentioned at the top of bars represent percentage staining of target proteins. IHC expression: Immunohistochemical expression; MIBC: Muscle invasive bladder cancer; NMIBC: Non-muscle invasive bladder cancer.

Figure 3 Representative images of immunohistochemical staining of marker protein in urinary bladder tumors at the magnification of 400×. A: Controls: a: Positive control for pS9GSK-3β showing strong membranous expression in normal urothelium; b: Positive control for β-catenin showing high membranous expression in normal urothelium; c: Negative control for Cyclin D1; d: Negative control for Snail; e: Negative control for Slug; B: Immunohistochemical (IHC) staining in non-muscle invasive bladder cancer cases; f: Nuclear/cytoplasmic expression of pS9GSK-3β; g: Nuclear expression of β-catenin (few tumor cells are circled); h: Cyclin D1 nuclear immunoexpression in 60%-70%; i: Snail nuclear immunopositivity in 80-90% cells; j: Slug nuclear expression in 60%-70% cells; C: IHC staining in muscle invasive bladder cancer cases; k: Loss of membranous pS9GSK-3β expression; l: Loss of β-catenin membranous expression; m: 100% nuclear staining for Cyclin D1 in urothelium; n: 100% nuclear immunoexpression of Snail in urothelium; o: 100% nuclear immunopositivity of Slug (Snail and Slug showed both nuclear and non-specific cytoplasmic staining, however, only nuclear expression was taken into account during analysis).

Figure 4 Kaplan-Meier overall survival curve analysis in patients with urothelial carcinoma of bladder.

Figure 5 Box plot representation of quantitative expression profile of β-catenin and its target genes (Cyclin D1, Snail and Slug) in a cohort of 90 urinary bladder tumors at transcriptome level in non-muscle invasive bladder cancer and muscle invasive bladder cancer, and low grade and high grade tumors. Boxes represent the 25^th to 75^th percentile, horizontal lines indicate median values, and stars represent the maximum value. A: Non-muscle invasive bladder cancer and muscle invasive bladder cancer; B: Low grade and high grade tumors. The Y axis was logarithmically scaled in all cases. MIBC: Muscle invasive bladder cancer; NMIBC: Non-muscle invasive bladder cancer.