Fig 2: Summary illustration of WWOX action upon DNA SSBInduction of SSBs activates ATR-CHK1 pathway. ATM is activated to mediate ATM-CHK2 response. ATM could phosphorylate ITCH that mediates WWOX ubiquitination and stabilization which together with p-ATM maintains efficient DDR and guard stability of the genome. Inhibition of ATM renders WWOX less active (not shown). Activation of WWOX acts in a feed-forward loop to maintain ATM efficient DDR (16) and possibly ATR checkpoint activation.

Fig 3: WWOX accumulation and ubiquitination in the nucleus following DNA SSBsA. MCF7 cells were untreated or treated with UVC as indicated in the figure for specific time points. Cells were then sub-fractioned into nuclear and cytoplasmic fractions, then analyzed by immunoblotting using ATM, p-ATM (Ser1981), KAP1, p-KAP1 (pThr824), CHK1, p-CHK1 (Ser296), lamin, and GAPDH. B. HEK293T cells were untreated or treated with HU as indicated in the figure for specific time points. Cells were then sub-fractioned in to nuclear and cytoplasmic parts, then analyzed by immunoblot using the indicated antibodies. C. DNA damage enhances WWOX ubiquitination. HEK293T cells were transfected with HA-UB, GST-WWOX, GST-K274R, and GST-WFPA plasmids, 24h later the cells were treated with IR or UVC or HU as indicated then analyzed by immunoblotting using antibodies against p-ATM (Ser1981), KAP1, p-KAP1 (pThr824), CHK1, p-CHK1 (Ser296), GST (WWOX) and GAPDH. GST pull-down was performed and the pull down complex was blotted using anti-HA, and anti GST.

Fig 4: WWOX modulation of ATR checkpoint is ATM-dependentA. WWOX associates with p-ATM following induction of DNA SSBs. HEK293 cells were transfected GST-WWOX. At 24 hours, cells were treated with IR (10Gy for an additional hour) or UV ((70 J/m2) or HU (2mM) for an additional 2 hours). Cells were then subjected to GST-pulldown and Lysates were blotted against ATM, p-ATM (Ser1981), KAP1, p-KAP1 (pThr824), CHK1, p-CHK1 (Ser296), GST (WWOX) and GAPDH. Pulled-down complexes were blotted with anti p-ATM (Ser1981) and anti-GST (WWOX). B. MCF7 cells were cultured in duplicates, first duplicate were left untreated ore treated with IR or UVC or HU, the second duplicate were treated first with ATM inhibitor KU-55933 for 48h then were treated as first duplicate as indicated in the figure. Whole cell lysates were analysed by western blot using antibodies against ATM, p-ATM, KAP1, p-KAP1, ITCH, CHK1, p-CHK1, WWOX, and GAPDH as loading control.

Fig 5: WWOX regulates DNA-damage response following SSBsA. MCF7-sh EV and MCF7-shWWOX cells were untreated or treated with UVC for the indicated time points. Whole cell lysate were analysed by immunoblot using specific antibody against CHK1, p-CHK1 (Ser296), WWOX, ?-H2AX (pSer139), and GAPDH. B. Immunoblot analysis of phospho-CHK1 activation in WWOX-overexpressing HeLa cells after HU treatment. HeLa-EV or HeLa-WWOX cells were treated with HU for the indicated times. Cell lysates were then probed with antibodies against CHK1, p-CHK1, WWOX and GAPDH. C. MCF7 depleted WWOX (MCF7-sh) cells were infected with lentiviral vector expressing EV, or WWOX or mutant WWOXK274R. Whole cell lysates were collected 1h following IR or 2h following UVC treatment then analyzed by immunoblotting using specific antibody against ATM, p-ATM (Ser1981), KAP1, p-KAP1 (pThr824), CHK1, p-CHK1 (Ser296), WWOX, and GAPDH.