Figure 3
Figure 3. ATL patient samples express high levels of β-catenin cotranscriptional genes, TCF-1 and LEF-1. (A) Western blot analysis of β-catenin, phosphorylated GSK3-β (Ser9), and STAT3 expression from total cellular extracts of 6 ATL patient samples. Extracts were normalized to actin expression. (B) Real-time PCR analysis of β-catenin expression from cDNA from ATL patients. Samples were normalized to GAPDH expression, and the fold change was calculated by comparing values with R.PBMCs’ normalized gene expression. (C) ATL cell lines were treated with MG132 (2.5 µM) or dimethyl sulfoxide control for 8 hours, followed by Western blot analysis on total cellular extract with β-catenin antibody. The upper band corresponds to mono-ubiquitinated β-catenin, the middle band corresponds to wild-type β-catenin, and the lower band corresponds to degraded β-catenin after MG132 treatment. (D) ATL cell lines were treated with 10 mM LiCl for 24 hours. Total cell lysates were normalized to actin expression before Western blot analysis with β-catenin or p-GSK3β (Ser9). (E) Real-time PCR expression of Wnt/β-catenin downstream target genes, c-myc, survivin, VEGF, c-jun, COX-2, and cyclin D1from total cDNA from ATL patients. Fold change was calculated by comparing values with Jurkat-normalized gene expression. The ATL patient cDNAs were additionally analyzed for Tax and p30 expression by qualitative PCR. Jurkat and LAF cDNA were used as negative and positive controls, respectively. (F) Real-time PCR for TCF-1, TCF-4, and LEF-1 from cDNA derived from ATL patients. Real-time PCR was performed in duplicate and samples were normalized to GAPDH expression. Fold change was calculated by comparing values with R.PBMC-normalized gene expression. (G) Cell cycle analysis of 1185 cells treated with or without IL-2, serum, and hydroxyurea after 48 hours. (H) Cells were treated as in (G) and analyzed by real-time PCR analysis. Real-time PCR was performed in duplicate and samples were normalized to GAPDH expression. Fold change was calculated by comparing values with nontreated 1185 cells’ normalized gene expression.

ATL patient samples express high levels of β-catenin cotranscriptional genes, TCF-1 and LEF-1. (A) Western blot analysis of β-catenin, phosphorylated GSK3-β (Ser9), and STAT3 expression from total cellular extracts of 6 ATL patient samples. Extracts were normalized to actin expression. (B) Real-time PCR analysis of β-catenin expression from cDNA from ATL patients. Samples were normalized to GAPDH expression, and the fold change was calculated by comparing values with R.PBMCs’ normalized gene expression. (C) ATL cell lines were treated with MG132 (2.5 µM) or dimethyl sulfoxide control for 8 hours, followed by Western blot analysis on total cellular extract with β-catenin antibody. The upper band corresponds to mono-ubiquitinated β-catenin, the middle band corresponds to wild-type β-catenin, and the lower band corresponds to degraded β-catenin after MG132 treatment. (D) ATL cell lines were treated with 10 mM LiCl for 24 hours. Total cell lysates were normalized to actin expression before Western blot analysis with β-catenin or p-GSK3β (Ser9). (E) Real-time PCR expression of Wnt/β-catenin downstream target genes, c-myc, survivin, VEGF, c-jun, COX-2, and cyclin D1from total cDNA from ATL patients. Fold change was calculated by comparing values with Jurkat-normalized gene expression. The ATL patient cDNAs were additionally analyzed for Tax and p30 expression by qualitative PCR. Jurkat and LAF cDNA were used as negative and positive controls, respectively. (F) Real-time PCR for TCF-1, TCF-4, and LEF-1 from cDNA derived from ATL patients. Real-time PCR was performed in duplicate and samples were normalized to GAPDH expression. Fold change was calculated by comparing values with R.PBMC-normalized gene expression. (G) Cell cycle analysis of 1185 cells treated with or without IL-2, serum, and hydroxyurea after 48 hours. (H) Cells were treated as in (G) and analyzed by real-time PCR analysis. Real-time PCR was performed in duplicate and samples were normalized to GAPDH expression. Fold change was calculated by comparing values with nontreated 1185 cells’ normalized gene expression.

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