Frame-shift mutations are exclusively detected in primary LICs with low CTNNA1 transcripts. (A) Quantitative real-time RT-PCR analysis of CTNNA1 transcripts in FACS-sorted LICs (CD34⁺CD38⁻CD123⁺Lin⁻) from MDS or AML patients. The down-regulation of CTNNA1 transcripts was arbitrarily defined as 60% reduction (dashed line) compared with normal HSCs and the control NB4 cell line. (B-C) Summary of frame-shift mutations detected in the PTEN (B) and CEBPA (C) genes. Note that these mutations were only identified in 6 of 8 patient's LICs expressing low levels of CTNNA1 transcripts. (D) Western blot analysis with protein lysates extracted from the MNCs of patients expressing low CTNNA1 and carrying frame-shift mutations in the PTEN gene (P-1, P-24, and P-26). Note the reduction of the p42/p30 ratio in patients P-1, P-24, and P-26 who had frame-shift mutations in the PTEN gene. #Wild-type PTEN protein that is probably expressed from the normal blood cells or blasts within the heterogeneous MNC mixture. (E) Western blot analysis with protein lysates extracted from MNCs of patient expressing low CTNNA1 and carrying N-terminal frame-shift mutations in the CEBPA gene (P-27). (F) Western blot analysis with protein lysates extracted from MNCs of patients expressing appropriate levels of CTNNA1 and carrying no mutations in either PTEN or CEBPA (P-25 and P-6). (G) The MNCs derived from patient P-1 were treated with rapamycin at the indicated concentrations for 5 days. Western blot analysis showed a dose-dependent increase in the p40/p30 C/EBPα ratio (left) and a coincident up-regulation of CTNNA1 transcripts as determined by quantitative real-time RT-PCR analysis (right). The Arabic numbers at the bottom of panels D through G denote the p42/p30 ratio. (H) ChIP analyses with the indicated antibodies in primary MNCs of patients expressing low (P-1, P-24, and P-35) and appropriate CTNNA1 levels (P-7, P-17, and P-22).