Genetools software program

Approximately 10⁴ cells were seeded in 100-mm dishes in duplicate. Subsequently, the cells were chronically treated with cisplatin or mitomycin C (MMC) for 10 days. The resulting colonies (anchorage on plates) were stained with 1% crystal violet (Sigma) for 2–3 min. After being washed with water several times to remove crystal violet solution. The colonies were then scanned with a scanner (Epson precision V33). The number of colonies was counted using the GeneTools software program (Syngene).

10⁶ cells were washed and resuspended in lysis buffer [50mM Tris (pH7.5), 150 mM NaCl, 1mM EDTA, 0.1% Triton X-100, protease inhibitor cocktail (MD Biol)]. After sonication, cell lysates were added with Laemmli sample buffer and boiled for 5 minutes. Samples were separated on a 10% SDS-PAGE and transferred to a PVDF membrane. Protein blots were probed using specific antibodies against γH2AX (05-636, Millipore), H2AX (ab11175, abcam), phospho-CHK1(S345) (2348, Cell Signaling Technology), CHK1 (sc-8408, Santa Cruz Biotechnology), phospho-CHK2 (T68) (2661, Cell Signaling Technology), CHK2 (sc-5278, Santa Cruz Biotechnology), Caspase 3 (9662, Cell Signaling Technology), and FOXM1 (sc-500, Santa Cruz Biotechnology). All images were acquired by the GeneGnome 5 (Bio Image, Syngene) and the γH2AX/H2AX, p-CHK1/CHK1, and p-CHK2/CHK2 ratios were quantitative by a GeneTools software program (Syngene). These data were repeated at least three times and similar trends were observed.

Approximately 10⁴ cells were seeded in 100-mm dishes in duplicate. Subsequently, cells were chronically treated with cisplatin, MMC, MMS, and 4NQO, and incubated for 10 days. The resulting colonies were stained with 1% crystal violet (Sigma). Colonies were counted using a GeneTools software program (Syngene).