Fig. 3: Wild type β-catenin translocates into the nucleus upon CHIR99021-mediated stabilization in melanoma cell cultures. | Oncogene

Fig. 3: Wild type β-catenin translocates into the nucleus upon CHIR99021-mediated stabilization in melanoma cell cultures.

From: Temporal activation of WNT/β-catenin signaling is sufficient to inhibit SOX10 expression and block melanoma growth

Fig. 3

a A representative western blot for the indicated proteins of cell lines carrying a stabilizing mutation in β-catenin (p.S37F) (Mel501, Mel888). M11 expresses wild type β-catenin and serves as a control. The human melanoma-derived cell lines were cultured in presence or absence of CHIR99021 for 24 h, 6 μM (n = 3). b Representative immunocytochemical stainings indicating the subcellular localization of β-catenin (in green) in a melanoma patient-derived cell culture carrying wild type β-catenin (M98) in presence of DMSO (left panel) or 6 µM CHIR99021 (right panel) for 24 h. Inserts show higher magnification of immunostainings. c Representative immunocytochemical stainings indicating the subcellular localization of β-catenin (in green) in a melanoma cell line harboring the stabilizing p.S37F mutation in β-catenin (Mel888) in presence of DMSO (left panel) or 6 µM CHIR99021 (right panel) for 24 h. Inserts show higher magnification of immunostainings. d β-catenin nuclear localization analysis of M98 upon DMSO or 24 h of 6 µM CHIR99021 treatment, respectively. e β-catenin nuclear localization analysis of Mel888 upon DMSO or 24 h of 6 µM CHIR99021 treatment, respectively. f Tissue Micro Array (TMA) of melanoma cell lines (upper panel) n = 66, primary melanoma (middle panel) n = 33, or metastases (lower panel) n = 121, showing the cytoplasmic localization of β-catenin. The TMAs were analyzed according to the expression level of β-catenin both nuclear and cytoplasmic. The TMAs were so divided into nuclear (N) and cytoplasmic (C) localization of β-catenin. The samples were divided into three groups with expression in <10%, (Cell lines: N = 98.5% C = 9,1%; Primary melanoma: N = 87.9%, C = 6.1%; Metastases: N = 92.6%, C = 8.3%) between 10% and 90% (Cell lines: N = 0%, C = 9,1%; Primary melanoma: N = 9,1% C = 12.1%; Metastases: N = 7.4% C = 24%) and more than 90% (Cell lines: N = 1.5%, C = 81.8%; Primary melanoma: N = 3.0% C = 81,8%; Metastases: N = 0% C = 67.8%). Asterisks indicate β-catenin negative nuclei, filled triangles β-catenin positive ones. g The quantification is shown in the respective analyzed tissues. Data represent mean ± s.d. Statistical significance was determined by unpaired, two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001 In each panel, n indicates the number of independent experiments performed.

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