HTLV-I cell lines express varying levels of β-catenin but remain responsive to Wnt stimulation. (A) Western blot analysis of β-catenin expression from total cellular extracts of PBMCs, Jurkat, and HTLV-I cell lines. Extracts were normalized to actin expression. (B) Real-time PCR was performed on β-catenin cotranscription factors, TCF-1, TCF-4, and LEF-1 from cDNA derived from HTLV-I cell lines. Real-time PCR was performed in duplicate and samples were normalized to GAPDH expression. Fold change was calculated by comparing values with resting PBMCs (R.PBMCs). (C) Real-time PCR expression of Wnt/β-catenin downstream target genes, c-myc, survivin, VEGF, c-jun, COX-2, and cyclin D1 from total cDNA from HTLV-I cell lines. Real-time PCR was performed in duplicate. Fold change was calculated by comparing values with Jurkat normalized gene expression. (D-F) PCR amplification of typical canonical Wnts (D), noncanonical Wnts (E), and their coreceptors (F) from cDNA derived from HTLV-I–transformed (MT2 and MT4) and immortalized (1185 and LAF) cell lines. Resting PBMCs (R.PBMCs) and the non–HTLV-I T-cell line, Jurkat, were used as controls. GAPDH amplification in nonsaturating conditions served as a control for the quality and quantity of the samples.