Extended Data Fig. 2: PARylation is an early and direct mediator of TRF1-FokI DSB formation.
From: Regulation of ALT-associated homology-directed repair by polyADP-ribosylation
a, [ADP/ATP] ratio in DMSO/PARGi treated WT-TRF1-FokI U2OS cells. Cells were treated with 1.5 mM/1 hr MMS. (b) Representative IF images and quantification of PAR at WT-TRF1-FokI DSBs after PARGi, PARGi-PARPi or TNKS1 knockdown. c, Representative IF images and quantification showing GFP-PARP1 localization in WT-TRF1-FokI cells treated with PARPi, PARGi or both. d, Representative IF images and quantification showing GFP-PARG localization in WT-TRF1-FokI U2OS cells. e, Left: Representative IF images and quantification of telomere foci size per cell in VA13 and Hela LT cells transfected with WT-TRF1-FokI from N = 2 independent assays. f, Representative stills of telomere (eGFP-TRF1) movement in U2OS cells treated with DMSO, PARPi or PARGi. Graph displays the cumulative Mean Squared Displacement (MSD) of 100 telomeres. g, Top: Schematic of DNA combing in G2-synchronized WT-TRF1-FokI cells treated with DMSO, PARPi, PARGi, or co-treated with PARPi and PARGi. Left: Quantification of telomeric fiber length of combined pulses. Right: Violin plot analysis of fork velocity. h, Graphs of CldU/IdU tract distribution of telomeric fibers in inhibitor treated U2OS-TRF1-FokI cells. n refers to the number of fibers containing TTAGGG signals analyzed from N = 2 independent assays. i, Representative IF images and quantification of BrdU synthesis at telomeres in the indicated cell lines after transfection with WT-TRF1-FokI and treated with PARGi or PARPi. j, Representative IF images and quantification of PCNA and (k) POLD3 localization at WT-TRF1-FokI telomeres treated with treated with DMSO, PARPi, PARGi, PARGi-Me or PARPi-PARGi. All inhibitor treatments, 5μM/4hrs unless otherwise indicated. All scale bars in IF panels=5μm. All graphed data in the figure are mean ± s.e.m. Unless otherwise stated, (n) is the number of cells analyzed and the number of independent assays (N) conducted is represented by black circles. Statistical significance was determined using one-way ANOVA. Digital images are deposited on Figshare. Graphed data is available as Source Data.
