R18 induces apoptosis by rescuing the nuclear localization of FOXO3a in cells expressing ZNF198-FGFR1. (A) Expression of constitutively activated 4ZF fusion tyrosine kinase results in cytoplasmic localization of FOXO3a. (B) Induced expression of nuclear-localized FOXO3aAAA mutant attenuates IL-3 independence of TonBaF cells transformed by 4ZF. (C) Expression of R18 dimer inhibits cytoplasmic sequestration of FOXO3a induced by 4ZF and rescues nuclear localization of FOXO3a. COS7 cells were cotransfected with 4ZF, CFP-tagged R18 dimer or mutant, and EGFP-FOXO3a or EGFP-FOXO3aAAA mutant. The molar ratio of DNA amount in the cotransfection experiment was 2:1:1 (4ZF/R18/FOXO3a) to ensure successful 4ZF transfection in cells that are both CFP and EGFP positive. (D) Expression of 4ZF abrogates FOXO3a-dependent transcription regulation. (Left) U2-OS cells were transiently cotransfected with the 3× IRS reporter plasmid encoding luciferase, pCMV-Renilla, and an increasing amount of plasmid encoding 4ZF. The luciferase activity was measured and normalized to Renilla luciferase activity. The fold of luciferase activity was normalized to the activity of cells in the absence of 4ZF transfection. Representative data are shown from repeated experiments. (Right) Decreased expression of Bim1 and p27^kip1 in TonBaF cells inducibly expressing 4ZF. Two isoforms of Bim1, L (long) and S (short), were detected. (E) Induced expression of R18 dimer rescued FOXO3a-dependent expression of Bim1 and p27^kip1 in TonBaF cells expressing 4ZF. The samples were analyzed by using a Zeiss LSM 510 META microscope with a 40×/1.30 objective lens. The pictures were taken by using a photo multipler tube (PMT) and analyzed by Adobe Photoshop 6.0 (Adobe, San Jose, CA).