Dotap reagent

For retroviral transduction, the PT67 NIH3T3-derived packaging cell line, expressing the 10A1 viral envelope, was purchased from CLONTECH Laboratories Inc. (Mountain View, CA). PT67 cells were cultured in DME (Irvine Scientific, Santa Ana, CA) supplemented with 10% FBS. For vector production PT67 cells at 70% confluence were incubated with a precipitated mixture of DOTAP reagent (Roche Pharmaceuticals, Basel, Switzerland), and saturating concentration of the pLNCX2DsRed2 plasmid for 18 h. For selections, cells were cultured in the presence of 200–1,000 μg/ml of G418 (Life Technologies, Grand Island, NY) in a step-wise manner. For RFP gene transduction, MCF-7_Her2.1 cells at approximately 60% confluence were incubated with a 1:1 precipitated mixture of retroviral supernatants of PT67 cells and F12K medium containing 7% FBS. After 72 h cells were harvested in trypsin/EDTA and sub-cultured into selective media containing 200 μg/ml of G418, which was gradually increased to 1,000 μg/ml in a step-wise manner. Clones expressing RFP were isolated with cloning cylinders (Bel-Art Products, Pequannock, NJ) by trypsin/EDTA and were amplified and transferred by conventional culture methods in the absence of G418.

Stable lines were generated by co-transfection of expression constructs with pCoHygro (Life Technologies) using the DOTAP Liposomal Transfection Reagent (Roche) and selection for hygromycin resistance at 100 ug/mL (Life Technologies). Transient transfections were conducted using the TransIT-2020 reagent (Mirus), and live imaging was performed 72 hours later. RNAi was performed with dsRNA generated from MEGAScript T7 Transcription kit (Life Technologies) and PCR products containing T7 promoter sequences and the target regions (Table S2). Tissue culture cells were treated with 5–10 ug of dsRNA for 5 days with DOTAP Reagent (Roche). Irradiation experiments were conducted by exposing cells to 5 Gy of X-rays from a 130 kv Faxitron TRX5200 and incubating them for various recovery times at 25°C. All results are based from at least two biological replicates.
Images were collected using an Applied Precision Deltavision microscope and analyzed using SoftworX software. FRAP experiments were conducted using a 488 nm QLM laser (Applied Precision) at 1 micron bleach size and 100% intensity, and images acquired using adaptive settings. Pre-bleach signal was set to 100% and used to normalize recovery signals.