Triamcinolone

RKO colorectal and M059K glioma cell lines were obtained from the American Type Culture Collection (ATCC). U2OS-DR cells, which contain a chromosomally integrated DR-GFP assay to measure HR repair, have been described previously (36 (link)). RKO, U2OS-DR and derivative cell lines were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) with l-glutamine containing 10% tetracyline-free (tet-free) fetal bovine serum (FBS; Clontech Laboratories and Atlanta Biologics). M059K cells were cultured in DMEM/F12 media supplemented with 10% tet-free FBS (Invitrogen), 0.05 mM non-essential amino acids, 0.5 mM sodium pyruvate and 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. All cells were maintained at 37°C with 5% CO₂. Tet-free serum was required to prevent DsRed gene expression in the EJ-RFP system from residual tetracycline found in most commercially available FBS preparations. For studies involving ddSceGR, cells were cultured in charcoal-stripped FBS (Invitrogen Corporation) to minimize the levels of endogenous glucocorticoids present in untreated FBS preparations. For the experiments involving serum deprivation, cells were washed two times in DMEM containing reduced FBS concentrations to remove residual serum, followed by replacement of the culture medium with DMEM containing 0.1% FBS.
The EJ-RFP assay plasmids were stably integrated into RKO, M059K and U2OS-DR cells using a fluorescence-activated cell sorting (FACS) enrichment strategy, which is described in detail in the ‘Results’ section. Briefly, plasmid transfection was performed by nucleofection in these cell lines (Lonza corporation), and blasticidin and zeocin antibiotics were used to select for cells containing integrated copies of the Sce-TetR and TO-DsRed vectors, respectively, as per the manufacturer’s specifications. At the indicated times, sterile doxycycline (Sigma-Aldrich Co., LLC) was added to cell cultures at a concentration of 1 µg/ml to induce de-repression of DsRed gene expression. Cells were subjected to sterile FACS-based enrichment using standard protocols at the MSKCC Flow Cytometry Core Facility. When indicated, single cell clones were isolated by limiting dilution in 96-well microplates, using previously established protocols (37 (link)). Individual clones were expanded and tested for activity by flow cytometry for the induction of DsRed gene expression and fluorescence after I-SceI plasmid transfection.
The ddSceGR plasmid was stably integrated into U2OS EJ-DR cells by nucleofection, followed by selection in G418 antibiotics for ∼2 weeks. Single cell clones again were isolated by limiting dilution, and individual clones were expanded, followed by testing for ligand-induced NHEJ and HR repair activity by flow cytometry. Ligand-induced DNA cleavage by ddSceGR was performed by adding the Shield1 and triamcinolone (TA) ligands at concentrations of 0.5–1 µM and 100 nM, respectively, to the cell cultures. Unless otherwise indicated, ligands were incubated in the cells for 24 h, followed by two washes with DMEM containing 10% FBS without ligands.