Figure 4: SIRT7 is transiently recruited to DNA-damage sites in a PARP1-dependent manner.

(a) Whole-cell extracts from HEK293T cells expressing vector or FLAG-SIRT7wt were subjected to affinity purification with anti-FLAG M2 affinity gel. The purified protein complex was resolved on SDS–polyacrylamide gel electrophoresis and silver stained. The bands were retrieved and analysed by mass spectrometry. (b) U2OS cells stably expressing GFP-SIRT7 were subjected to laser microirradiation using micropoint system and analysed for the accumulation of GFP-SIRT7 in DSBs by fluorescent microscopy (upper). Scale bar, 10 μm. The real-time recruitment of GFP-SIRT7 was also analysed in 30 independent cells (lower). Error bars indicate mean±s.e.m. (c) U2OS cells were pre-treated with PARP1 inhibitor PJ-34 or ATM inhibitor KU-55933 for 1 h and subjected to laser microirradiation and immunofluorescent analysis of SIRT7 and γH2AX at 5 min after microirradiation (upper). Scale bar, 10 μm. The expression of SIRT7 was analysed by western blotting (lower). (d) U2OS cells were exposed to 6 Gy of IR. Whole-cell lysate was immunoprecipitated with antibodies against SIRT7 followed by immunoblotting with the indicated antibodies.