Fig. 1: Identification of PARG as repair factor for TOP1-DPC by single-molecule tracking. | Nature Communications

Fig. 1: Identification of PARG as repair factor for TOP1-DPC by single-molecule tracking.

From: PARylation prevents the proteasomal degradation of topoisomerase I DNA-protein crosslinks and induces their deubiquitylation

Fig. 1

a Top panels: filming of TOP1-HaloTag single molecules in U2OS cells transfected with TOP1-HaloTag expression plasmid. The cells were divided into seven groups: no treatment, DMSO (2h), BTZ (10 μM, 2h), PARGi (10 μM, 2h),  CPT (100 μM, 2h), CPT + BTZ and CPT + PARGi. Middle panels: plots of tracks of TOP1-HaloTag single molecules in the top-panel films. The tracks were reconstructed in two dimensions by MATLAB analysis pipeline. The neighboring tracks are of different colors to distinguish one from the others. Bottom panels: count of jumps of TOP1-HaloTag single molecules derived from the top-panel films. The X-axis of the histogram is the jump distance of TOP1 single molecules in the top-panel films and the Y-axis is the count of the jumps. The bin size is 0.1 μm. For example, the “before treatment” sample has 4120 jumps whose distances are between 0 and 0.1 μm and 86 jumps whose distances are between 1 and 1.2 μm. The sample has a total of 19120 jumps that are the sum of the count of each bin. b Quantitation of TOP1-HaloTag single molecules shown in panel (a) using ThunderSTORM, an ImageJ plug-in. Top lines are maximum values, bottom lines are minimum values and middle lines are medians. Box limits indicate the range of the central 50%. Whiskers are upper and lower quartiles. n = 10 biologically independent samples. P value was calculated by paired Student’s t-test (two-tailed distribution). **: p = 0.0017. c Time-course of TOP1-HaloTag single molecules in U2OS cells treated with DMSO, CPT (100 μM), CPT (100 μM) + PARGi (10 μM) and CPT (100 μM) + BTZ (10 μM). Images were taken at the indicated time points. The scale bar represents 10 μm. d Quantitation of TOP1-HaloTag single molecules shown in panel (c). n = 2 independent experiments. Data are presented as mean values +/− standard deviation (SD). P value comparing TOP1 single molecules of CPT and CPT + PARGi was calculated by paired Student’s t-test (two-tailed distribution). *: p = 0.038.

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