Figure 1. (A) Inverse correlation between tyrosine⁷⁰⁵ phosphorylated STAT-3 (P-STAT-3) and SOCS-3 expression in human cholangiocarcinoma (CCA) tissue.

Immnohistochemistry for P-STAT-3 and SOCS-3 was performed using 26 surgically resected human CCA specimens (400X magnification). The percent of cells with cytoplasmic SOCS-3 was assessed, as was the percent of cells with nuclear P-STAT-3 staining. Samples are shown with positive P-STAT-3 (70–100% of cells) with low SOCS-3 staining (<30%), and separately with negative P-STAT-3 (<30%) and high SOCS-3 staining (>70%). (B) Quantitative analysis of SOCS-3 staining versus P-STAT-3 staining. Cells were examined and quantitated for P-STAT-3 nuclear staining and SOCS-3 cytoplasmic staining. At least 500 cells per specimen were counted. Each point represents the average value for one sample. Bars represent the mean SOCS-3 staining for all specimens within each subset of P-STAT-3 staining. (C) DNA methylation of the socs-3 promoter is associated with SOCS-3 silencing. Genomic DNA was extracted from either SOCS-3 (+) or SOCS-3 (−) CCA tissue procured by laser capture microdissection (LCM). Bisulfite-modified DNA was subjected to methylation-specific PCR as described in the Materials and Methods section using either a methylation- (M) or unmethylation- (U) specific primer set for the socs-3 promoter. (D, E) CpG island methylation of the socs-3 promoter is specific for CCA. Genomic DNA was extracted from paired tumor and non-tumor tissue (uninvolved bile duct epithelial cells and hepatocytes) procured by LCM. Cell-type specific genomic DNA was treated with bisulfite, subjected to methylation-specific PCR of the socs-3 promoter region (D) and sequenced (E). A total of 44 CpG sites located between nucleotides -678 and -216 of the socs-3 promoter were sequenced (E). The horizontal squares represent CpG islands while the vertical squares represent the individual 5 clones sequenced. Each black square represents a methylated cytosine residue within the CpG islands.