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. Author manuscript; available in PMC: 2015 Oct 24.
Published in final edited form as: Cell Rep. 2015 Oct 8;13(3):451–459. doi: 10.1016/j.celrep.2015.09.017

Figure 4. TIMELESS Stabilizes PARP1 Interaction with Its Substrates upon DNA Damage.

Figure 4

(A) HEK293T cells were transfected with either an EV or the indicated FLAG-tagged proteins. 24 hr post-transfection, WCEs were subjected to IP with α-FLAG resin and immunoblotted as indicated. Ex. Prot., exogenous proteins.

(B) The experiment was performed as in (A). Gels in the bottom panels were loaded as indicated in the upper panels, except that MWMs were omitted. Asterisks denote nonspecific bands.

(C) U2OS cells were transfected overnight with either an siRNA to TIMELESS or a control non-targeting siRNA (siCTRL). 24 hr later cells were then transfected with either an EV or FLAG-tagged PARP1. 24 hr post-transfection, WCEs were subjected to IP with α-FLAG resin and immunoblotted as indicated. White asterisks denote non-specific band. The experiment was repeated three times with identical results.

(D) PARP1 recruitment is impaired after laser microirradiation following knockdown of TIMELESS. U2OS cells stabling expressing PARP1-GFP were depleted of TIMELESS for three days using siRNA, and the kinetics of PARP1recruitment to DNA damage sites were assessed by live-cell imaging of laser-induced lesions. For each condition, n ≥ 15. SE is shown for each time point.

(E) KU80 recruitment is impaired after laser microirradiation following knockdown of TIMELESS. U2OS cells stabling expressing KU80-GFP were depleted of TIMELESS for 3 days using siRNA, and the kinetics of KU80 recruitment to DNA damage sites were assessed by live-cell imaging of laser-induced lesions. For each condition, n ≥ 20. SE is shown for each time point.

(F) Quantification of NHEJ measured as fold change in frequency of repair of EJ5-GFP, resulting in GFP-positive cells after expression of I-SceI in cells treated with the indicated siRNA oligos or drugs. Bar graphs represent the mean of eight independent experiments ± SD. p < 0.0001 (unpaired t test, using GraphPad software). Panel below shows efficiency of knockdown using western blot.

(G) Quantification of HR measured as fold change in frequency of repair of DR-GFP, resulting in GFP-positive cells after expression of I-SceI in cells treated with the indicated siRNA oligos or drugs. Bar graphs represent the mean of eight independent experiments ± SD. p < 0.0001 (unpaired t test, using GraphPad software). Panels below show efficiency of knockdowns using western blot.