Growth characteristics of NIH 3T3 cells overexpressing Wt and K199R HRI.
(A) Levels of HRI protein expression. NIH 3T3 cells overexpressing vector alone (lane 1), Wt HRI (lane 3), or K199R HRI (lane 2) were generated by infections with retroviruses as described in “Materials and methods.” Cytoplasmic extracts (20 μg) from these cells, as indicated in the Figure, were separated by 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed for HRI by Western blot analysis. Purified rabbit reticulocyte HRI (lane 4) and reticulocyte lysates (lane 5) were used as positive controls. (B) Growth curve using total cell numbers. NIH 3T3 cells expressing either the pLXSN retroviral vector alone, Wt HRI, or K199R HRI were seeded at equal densities of (2.5 × 105) per 100 mm. Cells expressing Wt HRI were washed 3 times with DMEM to remove the hemin supplemented in the medium and maintained in the culture medium without hemin supplement for 24 hours prior to seeding. Total numbers of cells at different times of culture were plotted. (C) Growth curve of viable cells. Cells were grown as described in panel B, and the cell viability was determined by staining with trypan blue. The number of trypan blue–negative cells was plotted relative to time in culture. (D) Phase contrast photographs. NIH 3T3 cells expressing vector, Wt, or K199R HRI at day 3 of growth as shown in panels B and C were photographed at 200 × magnification.
