T-REx Flp-In DLD-1 cells (provided by S. Taylor, University of Manchester, UK) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher) supplemented with 10% tetracycline-free fetal bovine serum (Omega Scientific) and 100 U/mL penicillin-streptomycin. Cells were maintained at 37°C under 5% CO2 and atmospheric oxygen. CENPA alleles were genetically modified in TIR1-expressing T-REx Flp-In DLD-1 cells26 by co-transfection with pcDNA3.1 plasmids (Invitrogen) encoding TAL effector nucleases, as previously described18 (link), and an EYFP-AID donor construct targeting the translation start codon of CENPA. Single EYFP+ cells were isolated by fluorescence-activated cell sorting (Sony SH800) and screened by immunoblotting and PCR for CENP-A–/EYFP-AID clones. CENP-AWT and CENP-AC-H3 rescue cDNAs were cloned into pcDNA5/FRT/TO plasmids and co-transfected with pOG44 into TIR1-DLD-1 CENP-A–/EYFP-AID cells using X-tremeGENE 9 (Roche). Cells that underwent stable Flp recombinase-mediated transgene integration at the FRT locus were selected with 100 μg/mL hygromycin (Thermo Fisher), and Y chromosome-positive clones were confirmed by FISH.
To generate stable cell lines expressing fluorescent reporters of interest, H2B-mRFP and mCherry-NLS-TagRFP (annotated as 2×RFP-NLS, a gift from E. Hatch and M. Hetzer, Salk Institute, USA) open reading frames were cloned into pBABE retroviral vectors and packaged in 293GP cells by co-transfection with pVSV-G using X-tremeGENE 9. Viral supernatants after 48- or 72-hour transfection were filtered (0.45 μm) and target cells were infected in the presence of 5 μg/mL polybrene (Santa Cruz) for ~16 hours. Fluorescent cells were isolated by fluorescence-activated cell sorting (Sony SH800).
Doxycycline and the auxin plant hormone indole-3-acetic acid (IAA) purchased from Sigma were dissolved in cell culture-grade water and used at 1 μg/mL and 500 μM, respectively. For cell cycle arrest experiments, 100 ng/mL nocodazole (Sigma) was used for mitotic arrest, 1 μM PD-0332991 (provided by S. Dowdy, UC San Diego, USA) was used for G1 arrest, and 10 μM RO-3306 (Sigma) was used for G2 arrest, all of which were dissolved in DMSO. The following DNA damage repair inhibitors were dissolved in DMSO and used at the indicated concentrations: 250 μM SCR7 (LIG4 inhibitor), 25 μM RI-1 (RAD51 inhibitor, both provided by A. Shiau, Ludwig Institute for Cancer Research, USA), and 10 μM NU7026 (DNA-PKcs inhibitor, Abcam).
All cell lines were tested for mycoplasma and confirmed free of contamination. The cell lines used in this study were not authenticated and are not found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI BioSample.
To generate stable cell lines expressing fluorescent reporters of interest, H2B-mRFP and mCherry-NLS-TagRFP (annotated as 2×RFP-NLS, a gift from E. Hatch and M. Hetzer, Salk Institute, USA) open reading frames were cloned into pBABE retroviral vectors and packaged in 293GP cells by co-transfection with pVSV-G using X-tremeGENE 9. Viral supernatants after 48- or 72-hour transfection were filtered (0.45 μm) and target cells were infected in the presence of 5 μg/mL polybrene (Santa Cruz) for ~16 hours. Fluorescent cells were isolated by fluorescence-activated cell sorting (Sony SH800).
Doxycycline and the auxin plant hormone indole-3-acetic acid (IAA) purchased from Sigma were dissolved in cell culture-grade water and used at 1 μg/mL and 500 μM, respectively. For cell cycle arrest experiments, 100 ng/mL nocodazole (Sigma) was used for mitotic arrest, 1 μM PD-0332991 (provided by S. Dowdy, UC San Diego, USA) was used for G1 arrest, and 10 μM RO-3306 (Sigma) was used for G2 arrest, all of which were dissolved in DMSO. The following DNA damage repair inhibitors were dissolved in DMSO and used at the indicated concentrations: 250 μM SCR7 (LIG4 inhibitor), 25 μM RI-1 (RAD51 inhibitor, both provided by A. Shiau, Ludwig Institute for Cancer Research, USA), and 10 μM NU7026 (DNA-PKcs inhibitor, Abcam).
All cell lines were tested for mycoplasma and confirmed free of contamination. The cell lines used in this study were not authenticated and are not found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI BioSample.
