Mycotest kit

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.

A549 and H1299 cells (classified as NSCLC cells) were collected from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium enriched with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin sulfate. The possible mycoplasma contamination was checked by Mycotest kit (Invitrogen, Chicago, IL, USA). The following antibodies were used in the study: pATM, FANCD2, Chk1, β-Actin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); Rad18 from Bethyl Laboratories, Inc. (Montgomery, TX, USA); pATM-Ser1981, pChk1-Ser317, pChk2, FANCJ, Bid, caspase-3, Snail and cyclin B are from Cell Signaling Technologies (Danvers, MA, USA); and γH2AX from Millipore company (Darmstadt, Germany); and E-cadherin from Becton Dickinson company (BD Biosciences, San Jose, CA, USA) Cell Signaling Technologies (The horseradish peroxidase (HRP)-conjugated secondary antibodies, such as anti-mouse IgG-Cy3 and anti-rabbit IgG, were procured from Molecular Probes (Santa Cruz, CA, USA).

The NSCLC cell lines A549 (human adenocarcinoma epithelial cell line) and H1299 (human were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% FBS, 100 μg/ml streptomycin sulfate and 100 U/ml penicillin. Normal human bronchial epithelial cells were grown in BEGM™ Bronchial Epithelial Cell Growth Medium as described previously [51 (link)]. Cells were routinely tested for mycoplasma contamination using Mycotest kit (Invitrogen) and cells within 10 passages were used in the experiments. AITC and PITC (Sigma, St. Louis, MO) stock solutions were prepared by dissolving in anhydrous DMSO and stored at −20°C. These stock solutions were further diluted to required concentration before adding to the cells. Antibodies to the following antigens used in this study include: ATR, ATM, Chk1, FANCD2 and GAPDH were from Santa Cruz Biotechnology, Inc.; Rad18, from Bethyl Laboratories, Inc.; phospho-ATM-Ser1981, phospho-Chk1 Ser-317 were Cell Signaling Technologies, γH2AX is from Millipore. The secondary antibodies like anti-mouse IgG-Cy3, anti-mouse IgG-FITC, anti-rabbit IgG-FITC were from Molecular Probes.