c-Myc T58A mutant conferred growth-factor independence on Ba/F3 cells.

(A) Schematic diagram of wild-type (c-Myc), T58A and In373 mutants of c-Myc. (B-H) Ba/F3 cells were infected with empty virus (−) or retrovirus encoding wild-type c-Myc (c-Myc) or two c-Myc mutants (T58A, In373). (B) Transduced cells were incubated without IL-3 (2 ng/mL) for 6 hr and whole cell lysates were immunoblotted (IB) with anti-c-Myc antibody or anti-β-actin antibody. (C, D) Transduced Ba/F3 cells were cultured with/without IL-3 (2 ng/mL) for 12 hr. (C) ODC mRNA was analyzed by quantitative real-time PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (D) Whole cell lysates were immunoblotted (IB) with anti-ODC antibody or anti-β-actin antibody. (E) Viable transduced Ba/F3 cells and VF/EpoR cells were counted in the presence and absence of IL-3 (2 ng/mL) for 3 days. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (F) Transduced Ba/F3 cells were left untreated or stimulated with IL-3 (2 ng/mL) for 24 hr. The viability of these cells was determined by the trypan blue exclusion method. Results are the mean ± S.D. of three independent experiments. (G) Transduced cells were cultured without IL-3 (2 ng/mL) for 24 hr. Cells were then fixed, treated with propidium iodide (PI) and subjected to FACS analysis. (H) Transduced Ba/F3 cells were cultured with/without IL-3 (2 ng/mL) for 24 hr. DNA was isolated from cells and subjected to agarose gel electrophoresis.