FIG. 9.

Overexpression of AMF1 affects cellular responses under etoposide treatment. (A) Overexpression of AMF1 in U2OS holds more cells in G₁ phase and inhibits etoposide-induced S-G₂ arrest and apoptosis. U2OS/AMF1 or U2OS/β-Gal cells were treated with 10 μM etoposide for 0, 24, and 48 h. Cells were harvested, processed, and subjected to flow cytometric analysis. Cell cycle stages are represented by the cellular DNA content, which was analyzed by PI staining and fluorescence-activated cell sorting. Boundaries for G₁ and S-G₂ phases are labeled on the top pair of graphs. (B) Western blotting analysis of AMF1, p53, and p21^WAF1/CIP1 in U2OS cells at 0, 4, 8, and 24 h after addition of 10 μM etoposide into culture medium. Protein concentrations in cell extracts were determined, and equal amounts were loaded in each lane as judged by the level of α-tubulin. Intensity of bands at 0 h shows the baseline level of each protein. (C) RT-PCR analysis of p21^WAF1/CIP1 transcripts in U2OS cells at 0, 4, 8, and 24 h after addition of 10 μM etoposide into culture medium. The 370-bp product was amplified using p21 oligonucleotides. As a control, the cDNA encoding GAPDH was amplified with specific oligonucleotides.