Forced expression of MEL1S blocks G-CSF–induced granulocytic differentiation. (A) Expression of MEL1 and MEL1S in retroviral infected L-G3 cell lines. Cell extracts from L-G3 cells with retrovirus containing Flag-tagged MEL1 or MEL1S were immunoprecipitated using anti-Flag M2 affinity gel followed by immunoblotting as described in “Materials and methods” (IP-Blot). Also, the extracts from COS7 cells with each CMV expression vector were used as controls for the immunoblotting analysis (Blot). Lanes 1, 2, and 3 represent L-G3 cells infected with a mock retrovirus (pLXSN), FLAG-tagged MEL1, or MEL1S, respectively. Lanes 4, 5, and 6 indicate control COS7 cells transfected with a mock vector (pCMV, lane 4), pCMV-MEL1 (lane 5), or pCMV-MEL1S (lane 6). As an internal control for immunoblot, a 40-kDa band of β-actin was shown at the bottom (lanes 4-6). Molecular weights are given in kilodaltons. (B) Growth curve of L-G3 cells infected by retrovirus with MEL1 or MEL1S. The indicated cells in the figures were subcloned by G418 selection after infection with the retrovirus containing FLAG-tagged MEL1 (LGMEL1 in the figure) or MEL1S (LGMEL1S). The mock construct (Mock) was used as a vector control. L-G3 cells (L-G3) were derived from parental cells as a control. Viable cells were counted by the trypan blue exclusion method at each time point. ○ or • indicates the culture conditions using medium with or without IL-3, respectively, and ▵ indicates medium with G-CSF. The error bars represent the standard deviations of 3 independent experiments. (C) Parental and infected L-G3 cells at IL-3, 4 or 6 days after the addition of G-CSF. The cells were stained with May-Grünwald-Giemsa solution. The parental L-G3, mock, and LGMEL1 cells show a change in the nuclear-cytoplasmic ratio and nuclear indentations signifying differentiation in response to G-CSF. In comparison, LGMEL1S cells expanded in the presence of G-CSF with an immature nuclear morphology. Original magnification, × 100.