Figure 3(A) U2OS stably expressing mCherry-LacR-FokI were transfected with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield1 and 4-OHT. Immunofluorescence against the indicated DNA repair proteins was subsequently performed to monitor their accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 100 cells per condition were counted (n=3 biological replicates). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.005, **P≤0.0005. (B) U2OS (left panel), RPE1-hTERT (middle panel), and HeLa cells (right panel) were monitored for their capacity to form IR-induced BRCA1 foci. For each cell line, the following conditions were used: U2OS cells were transfected with a nontargeting siRNA (siCtrl) or an siRNA targeting human POGZ (siPOGZ-1 or -2); RPE1-hTERT cells were transduced a control shRNA (shCtrl) or a shRNA directed against human POGZ (shPOGZ-1 or -2); HeLa cells were expressing a non-targeting sgRNA (sgCtrl) or a sgRNA targeting human POGZ and sub-cloned (POGZ∆-1 or -2). Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post-exposure to IR. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of ΒRCA1 foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.005, **P<0.001. (C) Representative images used for quantification in (B). Scale bar = 5 μm. (D) Schematic diagram outlining the different domains of human POGZ. Each structural domain and interacting partners are indicated. (E) U2OS stably expressing mCherry-LacR-FokI were transduced with the indicated shRNA and subsequently transfected with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield-1 and 4-OHT. Immunofluorescence against BRCA1 was subsequently performed to monitor its accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 50 cells per condition were counted. Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.0001. (F) U2OS cells containing the DR-GFP reporter construct were transfected with the indicated siRNA. 24h post-transfection. Cells were transfected with the I-SceI expression plasmid or an empty vector (EV), and the GFP+ population was analyzed 48h post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition provided with I-SceI (siCtrl, I-SceI). Data are represented as a bar graph showing the relative mean ± SEM, each replicate being representing as a round symbol (n=3 biological replicates). Significance was determined by one-way ANOVA followed by a Dunnett's test. *P≤0.0001. (G) U2OS stably expressing mCherry-LacR-FokI were transduced with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield-1 and 4-OHT. Immunofluorescence against the indicated HP1 isoform was subsequently performed to monitor its respective accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 75 cells per condition were counted. Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.005, **P<0.0001.