Extended Data Fig. 3: PARP1 activity controls the recruitment of PDHE1α to sites of DNA damage.
From: PARylated PDHE1α generates acetyl-CoA for local chromatin acetylation and DNA damage repair
a, Co-Immunoprecipitation (Co-IP) analysis of the interaction of PDHE1α and PARP1 in Flag-PARP1 and HA-PDHE1α overexpressed cells. b, Immunoprecipitation analysis of the endogeneous interaction between PDHE1α and PARP1 in the whole cell lysate fraction extracted from HeLa cells. c, Western blot of the PDHE1α signal at chromatin fraction from HeLa cells treated with different doses of olaparib. PAR signaling was used as a positive control of olaparib treatment efficiency. d, Western blot showing that treatment with the PARP1 inhibitors, olaparib and AG14361, had no effect on total protein levels in whole cell lysates extracted from HeLa cells. e, Western blot showing the PDHE1α signal in the chromatin fraction from MEF-WT and MEF-PARP1−/− cells treated with etoposide (10 μM) for 1 h or IR (3 Gy). f, MEF PARP1−/− cells were rescued with Flag-vector and Flag-PARP1 constructs. Western blot showing PDHE1α recruitment in chromatin fractions from HeLa cells following DNA damage. g, Western blot showing the PDHE1α signal in the chromatin fraction from HeLa cells transfected with NC siRNA and siRNA targeting PARP1 and treated with 3 Gy IR. h-i, Dynamics of PDHE1α accumulation at DNA damage sites monitored by laser micro-irradiation-coupled live-cell imaging of HeLa cells stably expressing shNC and shPARP1 constructs (h) and PARP1 inhibitor treated cells (i). GFP signal accumulation intensity at DSB stripes was quantified from three independent replicates (n = 20 for each group), the curve shows the mean ± s.e.m. Scale bars, 5 μm.
