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. 2009 Mar 4;284(33):22155–22165. doi: 10.1074/jbc.M808182200

FIGURE 4.

FIGURE 4.

PHLPP1 and PHLPP2 expression were induced by Abl kinase inhibitor or knockdown of Bcr-Abl in CML progenitor cells. A, the purified ALDHhi cells from one CML patient were isolated and cultured in semisolid methylcellulose medium (MethoCult H4435) as described under “Experimental Procedures” (upper right). The ALDHhi cells were transfected with control siRNA and Bcr-Abl siRNA, stained with phycoerythrin as described under “Experimental Procedures,” and viewed under a confocal microscope (upper left). The ALDHhi cells, which were untransfected or transfected with Bcr-Abl siRNA, were treated with Abl kinase inhibitors for 14–17 days. The numbers of CFU-GEMM, CFU-GM, and BFU-E were then counted. The cells were untreated, treated with Abl kinase inhibitors for 24 h, or transfected with Bcr-Abl siRNA after 5 days. When untreated, the mean number of CFU-GEMM was 36 (range, 32–40). When treated with STI571, AMN107, BMS354825, or Bcr-Abl siRNA transfection, the mean numbers of CFU-GEMM were 3.5 (2–5), 4 (2–6), 1 (0–2), and 1 (0–2), respectively. When untreated, the numbers of CFU-GM were 267 (218–316). When treated with STI571, AMN107, BMS354825, or Bcr-Abl siRNA transfection, the mean numbers of CFU-GEMM were 36.5 (31–42), 48.5 (42–55), 46.5 (39–54), and 43.0 (37–49), respectively. When untreated, the numbers of BFU-E were 147 (125–169). When treated with STI571, AMN107, BMS354825, or Bcr-Abl siRNA transfection, the mean numbers of CFU-GEMM were 32.5 (28–37), 26.5 (22–31), 20.0 (14–26), and 18.0 (11–25), respectively. CFU-GEMM, CFU-GM, and BFU-E were enumerated after 14–17 days of in vitro culture. These data represent the number of individual colonies produced per 1000 cells plated for doses ranging from 2 × 102 to 2 × 103 ALDHhi cells. The rate of the progenitor cells was evaluated as the percentage of the corresponding control (bottom panel). Each bar represents the mean of S.D. of three independent experiments. B, the relative expression levels of PHLPP1 and PHLPP2 mRNA of CFU-GEMM, CFU-GM, and BFU-E derived from Bcr-Abl siRNA-transfected ALDHhi cells were assessed after 14–17 days of treatment with the Abl kinase inhibitors. For the analysis of PHLPP1 and PHLPP2 expression, quantitative RT-PCR was performed relative to the G3PDH gene. Each bar represents the mean of S.D. of three independent experiments. Fourteen days after transfection with Bcr-Abl siRNA, the mRNA expression of Bcr-Abl was assessed by RT-PCR in CFU-GEMM, CFU-GM, and BFU-E (bottom panels). RT-PCR results are representative of three independent experiments. FITC, fluorescein isothiocyanate.