Thymidine induces RS in cultured dCK^−/− thymocytes. (A) Relative thymidine abundance in various tissues from C57BL/6 mice as determined by LC-MS/MS. Concentrations given in μmol/g of whole tissue. Data are means ±SEM; n = 5/tissue type. (B) Concentrations of thymidine (in μM) from C57BL/6 plasma and from standard OP9-DL1 culture medium, as determined by LC-MS/MS. Data are means ±SEM; Plasma, n = 7; Media, n = 3. (C) Representative CellTrace Violet (CTV) dye dilution curves from WT, dCK^−/−, TK1^−/−, and DKO DN3a thymocytes cultured on OP9-DL1 stroma for 4 d without thymidine supplementation in the medium. Red numbers above the distinct CTV peaks reflect the total number of completed cell divisions executed by the thymocyte populations. WT, dCK^−/−, and TK1^−/−, n = 4; DKO, n = 1. (D) CTV dye dilution curves after 4 d of culturing in the presence of 20 and 100 µM thymidine added to the culture medium. WT, dCK^−/−, and TK1^−/−, n = 4; DKO, n = 1. (E) Percent of live cells that are pH2A.X positive in WT (black circles), dCK^−/− (blue squares), TK1^−/− (red up triangles), and DKO (green down triangles) DN3b thymocytes after 48 h of stimulation in increasing concentrations of thymidine. WT, dCK^−/−, and TK1^−/−, n = 2; DKO, n = 1. *, Cessation of measurements of pH2A.X levels caused by massive cell death (>80% sub-G₁ staining; not depicted) induced by exposure of dCK^−/− cells to concentrations of thymidine equal to or greater than 50 µM. (F) pH2A.X expression in WT, dCK^−/−, TK1^−/−, and DKO DN3b thymocytes after 12 h of exposure to increasing concentrations of hydroxyurea 36 h after plating on OP9-DL1 stroma. WT, dCK^−/−, and TK1^−/−, n = 2; DKO, n = 1.