Figure 6.
Effect of Id1 on myeloid leukemic cell line proliferation and expression of cell cycle regulators in Id1 overexpressing bone marrow cells (BMC). (a) Mo7e cells were electroporated with either Id1 siRNA oligo or control oligo with designated concentration in 100 µl buffer. Immediately following electroporation, cells were seeded at a concentration of 1 × 106 cells per ml in culture containing RPMI 1640, 10% fetal calf serum (FCS), 20 ng/ml granulocyte monocyte colony-stimulating factor (GM-CSF) and 100 ng/ml stem cell factor (SCF). Id1 protein knockdown was confirmed by western blotting 48 h after electroporation. (b) The viable cell number was determined each day over a 3-day period by trypan blue exclusion (*P < 0.01). (c) Protein levels of cell cycle regulators in 5FU-Id1 BMC were measured by western blotting. 5FU-treated Id1 overexpressing BMC were maintained with CYTOKINES (SCF + Flt3-L + IL-6 + TPO) over 9 months (M), and cells were harvested at the times indicated. Western blot of the total cellular extracts from the cells was probed with p15, p16, p19 and p21 antibodies. The blot was stripped and reprobed with anti-actin antibody to verify equal protein loading.