FIG. 1.

Inducible expression of Gadd45a results in apoptosis in HeLa cells. (A) a HeLa Gadd45a-inducible cell line was established as described in Materials and Methods. Cells were placed in 100-mm dishes at a density of 4 × 10⁵ and grown in DMEM containing tetracycline at a concentration of 2 μg/ml. After withdrawal of the tetracycline, the cells were collected at the indicated time points for preparation of cellular protein. A total of 100 μg of whole-cell protein was used for immunoblotting analysis with anti-Gadd45a antibody. In addition, Gadd45a protein levels in HeLa cells treated with UV at a dose of 10 J/m² or MMS at a concentration of 100 μg/ml were examined. As a loading control, anti-actin antibody was included. (B) HeLa Gadd45a-inducible cells were seeded at a density of 1,000 cells per 100-mm dish and grown in medium containing 2 μg of tetracycline/ml. After 16 h, the medium was removed, the plates were washed three times with PBS, and then fresh medium containing no tetracycline was added to the plates. The cells were fixed and stained at 14 days and scored for colonies containing at least 50 cells. The experiments were performed four times, and only representative results are shown here. (C and D) After inducible expression of Gadd45a protein, cells were collected at the indicated time points for the assay of cellular DNA fragmentation (C) or for measurements of cleavages of PARP and caspase-3 (D).