STAT3-dependent growth of Ba/F3 cells is inhibited by PML and augmented by PML/RARα.

PML and PML/RARα were introduced into Ba/F3 cells expressing G-CSF-R/gp130. According to the expression levels of the transgene, 5 clones in which PML or PML/RARα was expressed efficiently were selected from the respective transfectants. These 5 clones were mixed with an equal ratio to prepare the mixed clones from the respective transfectants. (A) The protein expression level of PML and PML/RARα in a representative clone of each transfectant. (B-C) The responses to IL-3 and G-CSF in a representative clone of each transfectant. After IL-3 deprivation, the cells were stimulated with 30 ng/mL G-CSF (B) or 1 ng/mL IL-3 (C). Induction levels of STAT3-responsive (B) or STAT5-responsive (C) genes were examined by Northern blot analysis. (D-I) The growth characteristics of respective transfectants were examined in 5 single-cell clones and the mixed clone. All of 5 clones and the mixed clone showed similar growth responses to IL-3 or G-CSF. ● indicates mock; ▪ PML; ▴ PML/RARα. The data obtained from the mixed clones were shown as the means ± SDs of triplicate cultures. (D-E) Dose responses to IL-3 and G-CSF were examined by MTT assays. O.D. indicates optical density. (F-I) Total cell number was counted under the culture with 1 ng/mL IL-3 (F), 30 ng/mL (G), 1 ng/mL (H), or 0.1 ng/mL (I) G-CSF.