Figure 5.

DNA damage signaling is enhanced in Rad50^S/S Atm^-/- cells compared with Atm^-/- cells. (A) SQ/TQ foci formation in wild-type (WT), Atm^-/-, Rad50^S/S, and Rad50^S/S Atm^-/- primary ear fibroblasts was assessed by immunofluorescence. Primary ear fibroblasts were harvested 30 min after mock treatment (white bars) or 4 Gy of IR (gray bars). Two-hundred-fifty cells were counted for presence or absence of SQ/TQ foci. A cell is considered positive when it shows ≥10 foci. Data are from three experiments. (B) SQ/TQ foci from mock-treated or 4 Gy-irradiated primary ear fibroblasts. (C) Primary ear fibroblasts were pretreated for 1 h with caffeine (20 mM) or vehicle and harvested 30 min after treatment with 4 Gy IR as indicated. For each treatment, 250 cells were counted for presence or absence of SQ/TQ foci. Data are from two experiments run in duplicate. (D) Extracts from primary MEF cultures prepared after mock treatment (-); 1 h after 1 μM CPT, 2 mM HU, or 20 J/m² UV; or 10 min after 5 Gy of IR treatment were sequentially immunoblotted with γ-H2AX and Actin (loading control) antisera. “Short” and “Long” represent short and long exposure, respectively.