Skip to main content
. 2010 Jan 29;285(17):12935–12947. doi: 10.1074/jbc.M109.071696

FIGURE 5.

FIGURE 5.

WIP1 inhibits damage-induced focus formation. A, overexpression of WIP1 inhibits IR-induced damage foci in HeLa cells. HeLa cells were mock-transfected or transfected with either wild-type or D314A mutant WIP1-FLAG expression plasmid. At 24 h after transfection, cells were irradiated with 5-Gy IR. After 30 min, cells were fixed and immunostained with anti-γ-H2AX (red fluorescence) and anti-FLAG antibodies (green fluorescence) to assess damage-induced foci formation. 4′,6-Diamidino-2-phenylindole staining (DAPI, blue fluorescence) was used to identify nuclei. B, quantitative analysis of γ-H2AX-asociated damage foci in IR-treated HeLa cells with or without overexpressed WIP1. The fluorescence intensity values of γ-H2AX from >1300 independent cells from the mock-transfected and WIP1-transfected cells were measured by high throughput microscopic imaging and plotted. Error bars represent the standard error. C, fluorescence intensity of γ-H2AX-associated damage foci is inversely correlated with WIP1 fluorescence intensity. At 30-min post-IR, individual WIP1-transfected HeLa cells were subdivided into low (n = 138), medium (n = 406), or high (n = 181) WIP1-expressing cells based on high throughput quantitative fluorescence imaging. The fluorescence intensity values for γ-H2AX for each cell were also measured, and mean γ-H2AX fluorescence for each of the three WIP1 expression categories was plotted. Error bars represent the standard error (Student's t test; p < 10−10 between Low and Med and p < 10−40 between Med and High). D, knockdown of WIP1 in MCF7 cells results in increased γ-H2AX-associated damage foci. MCF7 cells were transfected with negative control siRNA or WIP1 siRNA. At 24 h after transfection, cells were irradiated with 5-Gy IR, fixed at 3 h after irradiation, and immunostained with anti-γ-H2AX (red fluorescence) and anti-WIP1 antibodies (green fluorescence). E, WIP1 inhibition γ-H2AX-associated damage foci is ATM-independent. GM09607 (ATM null) fibroblasts were transfected with WIP1-FLAG expression plasmid. After 24 h, cells were irradiated with 5-Gy IR, fixed 30 min after irradiation and then immunostained with the indicated antibodies.