Fig. 3: Evaluation and quantification of foci upon PARP15 inhibitor treatment.
From: Screening assay to monitor mono-ADP-ribosylhydrolase activity of viral macrodomains in cells
a Schematic representation of the assay when used to evaluate small molecule inhibitors targeting PARP15 (created in BioRender. Verheugd, P. (2026) https://BioRender.com/a35t588). MARylation-dependent PARP15.1 foci are resolved upon addition of OUL232 or OUL243, resulting in a shift from foci formation to a uniform, spread signal distribution. b–e HeLa-Flp-In TRex-GFP-PARP15.1-T2A-3xFLAG-NBGFP cells were treated with 1 µg/ml doxycycline to induce protein expression. Additionally, the cells were treated or not with the indicated amounts of compounds OUL232 or OUL243 overnight. DMSO served as the vehicle control. One hour prior to analysis by confocal microscopy, SpyDNA was added to stain nuclei. Live-cell imaging was performed using confocal microscopy. b Representative confocal microscopy images showing GFP-PARP15.1 (magenta), SpyDNA (blue) or the merged images. c Schematic representation of the established CellProfiler pipeline. d, e Images obtained from confocal microscopy were analyzed with CellProfiler. d Data were plotted with ggplot in R to indicate percentages of cells showing either nuclear foci (blue), uniformly distributed, spread signal (black), or a combination of both (yellow) for the indicated treatments. e Boxplot overlaid with individual values illustrating the number of foci per nucleus for the indicated treatments, differentiating between foci only (blue), spread signal (black) or a combination of both (yellow). Outliers were removed by the IQR method and p-values were calculated using pairwise Wilcoxon test, adjusted by false discovery rate applying the rstatix package in R. (**p ≤ 0.01; ****p ≤ 0.0001; ns, not significant; of 2–5 biological replicates, a total of 182 (control (n = 4)); 103 and 56 (DMSO 0.05% (n = 3) and 0.1% (n = 2), respectively); 77, 95 and 147 (OUL232 1.25 µM (n = 3), 2.5 µM (n = 4) and 5 µM (n = 5), respectively); 103 and 59 (OUL243 5 µM (n = 3) and 10 µM (n = 2), respectively) nuclei with GFP signal were evaluated).
