PMC:6145447 / 31026-32548 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/6145447","sourcedb":"PMC","sourceid":"6145447","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6145447","text":"I-SceI-induced DSB repair assays\nU2OS cell lines which harbor chromosomally-integrated I-SceI-based GFP reporter substrates were used to monitor the efficiency of HDR, SSA, MMEJ, and c-NHEJ repair processes. The DR-GFP, SA-GFP, EJ2-GFP, and EJ5-GFP cell lines (generous gifts from Dr. J. Stark, City of Hope Cancer Center) were transfected with 1 μg of I-SceI expressing vector (FuGENE 6 transfection reagent (Promega), Opti-MEM (Gibco)) in the media supplemented with DMSO or one of the following compounds: CK-666 (Sigma Aldrich: SML-006, 50 μM), CK-548 (Sigma Aldrich: C7499, 25 μM), CK-689 (Sigma Aldrich: 18251750 μM), SMIFH2 (Sigma Aldrich: S4826, 5 μM), or wiskostatin (Sigma Aldrich: W2270, 3 μM). The I-SceI expressing vector was a gift of Dr. Richard Baer. For the FLAG actin construct overexpression experiments, DR-GFP cells were transfected with FLAG-WTactin-NLS or FLAG-R62Dactin-NLS for 48 hr prior to transfection with I-SceI in media supplemented with DMSO or CK-666. For the mCherry actin construct overexpression experiments, DR-GFP cells were co-transfected with I-SceI and mCherry-WTactin, mCherry-WTactin-NLS, mCherry-R62Dactin, or mCherry-R62Dactin-NLS. Cells were harvested 48 hr post I-SceI transfection and % GFP+ events were counted by FACS (10,000 cells per biological replicate). For all conditions, parallel transfection with 1 μg of pEGFP-N3 vector (Clontech) was used to determine transfection efficiency. Percentage refers to the number of GFP+ cells divided by the number of pEGFP+ cells.","divisions":[{"label":"Title","span":{"begin":0,"end":32}}],"tracks":[]}