Characterization of DNA Repair in Cancer Cell Lines

H1299 (human non-small cell lung carcinoma) and A2780 (human ovarian cancer) were obtained from ATCC, and HCT116 and HCT116 RAD18^−/− (human colon cancer) cells were from Dr. Tadahiro Shiomi [27 (link)] and as used earlier [29 (link)]. Cells were cultured in Dulbecco's Modification of Eagle's Medium (DMEM) (Mediatech) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 1x Penicillin/Streptomycin sulfate (Gibco) [57 (link)]. Cells were routinely tested for mycoplasma contamination using Mycotest kit (Invitrogen) and were used prior to ten passages. CPT (Sigma) was used at concentrations and time periods indicated. Antibodies against the following targets were used: FANCD2, Rad51, 53BP1, GAPDH (all from Santa Cruz Biotechnology) BRCA2, RAD18 (Bethyl Laboratories) and γH2AX (Millipore). To express wild-type or the RING mutant RAD18, HCT116−/− cells were transfected with pcDNAmycRAD18 and pcDNARAD18 C28F, respectively.

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Tripathi K., Mani C., Clark D.W, & Palle K. (2016). Rad18 is required for functional interactions between FANCD2, BRCA2, and Rad51 to repair DNA topoisomerase 1-poisons induced lesions and promote fork recovery. Oncotarget, 7(11), 12537-12553.

Publication 2016

FBS Penicillin/Streptomycin sulfate Mycotest kit FANCD2 Rad51 GAPDH BRCA2 RAD18 γH2AX

53bp1 Antibodies Brca2 Cells Colon cancer Cultured medium Fancd2 Fetal bovine serum Gapdh Hct116 cells Human Mycoplasma Non small cell lung carcinoma Ovarian cancer Penicillin Rad18 Streptomycin sulfate

Corresponding Organization : University of South Alabama

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Variable analysis

independent variables

CPT (Camptothecin) concentrations and time periods
Expression of wild-type or RING mutant Rad18 in HCT116 Rad18-/- cells

dependent variables

FANCD2, Rad51, 53BP1, BRCA2, RAD18, and γH2AX protein levels

control variables

Cell lines: H1299 (human non-small cell lung carcinoma), A2780 (human ovarian cancer), HCT116 and HCT116 Rad18-/- (human colon cancer)
Cell culture media: Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1x Penicillin/Streptomycin sulfate
Mycoplasma contamination testing using Mycotest kit (Invitrogen)
Cell passage number: used prior to ten passages

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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