B. CDKL5 is recruited to FokI-induced DSBs. GFP alone (top panel) or GFP NLS-CDKL5 (middle and bottom panels) were stably expressed in U-2-OS 265 DSB reporter cells. Cells were mock treated or treated with 1 µg/ml doxycycline for 3 h to induce transcription of the reporter gene. An hour before DSB induction cells were treated with 0.3 µM PARG and 10 µM ATM inhibitor. Site specific DSBs were induced by treating the cells with 4-OHT and Sheild1 ligand. Cells were live imaged at 37 °C, between 15-25 minutes following DSB induction. Representative image showing the recruitment of GFP NLS-CDKL5 to FokI induced DSBs upstream of transcriptionally active (middle) but not the inactive (bottom) MS2 gene. GFP alone is used a control (top). White arrowheads mark the location of the mCherry-FokI upstream of the MS2 reporter cassette. Images are representative of multiple technical replicates of three independent experiments. Scale bar is 10 μm 