HEK 293T, Rad18−/− HCT116 (Shiomi et al., 2007 (link)), and U2OS cells were grown in high glucose DMEM (Gibco) with penicillin/streptomycin (Gibco), glutamine (Gibco), and 10% fetal bovine serum. Cells were damaged with MMS (Sigma-Aldrich), mitomycin C (Sigma-Aldrich), EMS (Sigma-Aldrich), 4-nitroquinoline 1-oxide (Sigma-Aldrich), aphidicolin (Sigma-Aldrich), camptothecin (Sigma-Aldrich), actinomycin D (EMD Millipore), etoposide (Sigma-Aldrich), hydrogen peroxide (Sigma-Aldrich), or cis-platinum (Sigma-Aldrich) at the indicated concentrations and times. Cells were also treated with ATR inhibitor (ATR-45; Charrier et al., 2011 (link)), ATM inhibitor (KU55933; Abcam), MG132 (Sigma-Aldrich), or LLnL (EMD Millipore) at the indicated concentrations and times.
Protocol detail
DNA Damage Response Assay in Cell Lines
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high glucose DMEM
penicillin/streptomycin
glutamine
mitomycin C
4-nitroquinoline 1-oxide
aphidicolin
camptothecin
actinomycin D
etoposide
hydrogen peroxide
cis-platinum
MG132
4 nitroquinoline 1 oxide
Actinomycin d
Aphidicolin
Camptothecin
Cells
Cis platinum
Etoposide
Fetal bovine serum
Glucose
Glutamine
Hydrogen peroxide
Ku55933
Mg132
Mitomycin c
Penicillin
Rad18
Streptomycin
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Variable analysis
independent variables
- MMS (Sigma-Aldrich)
- Mitomycin C (Sigma-Aldrich)
- EMS (Sigma-Aldrich)
- 4-nitroquinoline 1-oxide (Sigma-Aldrich)
- Aphidicolin (Sigma-Aldrich)
- Camptothecin (Sigma-Aldrich)
- Actinomycin D (EMD Millipore)
- Etoposide (Sigma-Aldrich)
- Hydrogen peroxide (Sigma-Aldrich)
- Cis-platinum (Sigma-Aldrich)
- ATR inhibitor (ATR-45; Charrier et al., 2011)
- ATM inhibitor (KU55933; Abcam)
- MG132 (Sigma-Aldrich)
- LLnL (EMD Millipore)
- Not explicitly mentioned
- HEK 293T cells
- Rad18^-/- HCT116 cells (Shiomi et al., 2007)
- U2OS cells
- High glucose DMEM (Gibco) with penicillin/streptomycin (Gibco), glutamine (Gibco), and 10% fetal bovine serum
- Positive control: Not specified
- Negative control: Not specified
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