Science - USA (2022-04-29)

SCIENCEscience.org 29 APRIL 2022¥VOL 376 ISSUE 6592 479

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Alkaline Tail Moment

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CAD

Ponceau

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AT R i

Caspase

ATM/ATR

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ICAD-L S107 D117 D224 S257

pS257 ICAD

GFP

pS107 ICAD

GFPWTDSA GFPWT DSA

IR (8Gy) 24h

Pulldown: GFP-trap

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-ICAD

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ICAD KO

U2OS :: GFP

ICAD KO :: GFP

ICAD KO :: WTICADICAD KO :: DSAICAD

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Fig. 3. ATM/ATR signaling to ICAD regulates CAD-induced DNA breaks
after IR.(A) U2OS cells expressing mCherry-ICAD and GFP-CAD were
microirradiated and imaged every minute for 25 min. Scale bar, 10mm. Data
are means ± SEM; cells per biological replicate,n= 6; biological replicates,
N= 3. (B) Recruitment of CAD and ICAD to microirradiated laser stripes after
inhibition of ATR. Conditions as in (A), except that ATRi AZ20 was added
20 min before microirradiation. RFI, SEM, replicates, andn/Nare the same as
in (A). (C) ATR inhibition (AZ20) for the indicated times prior to collection
affects chromatin recruitment of CAD at 24 hours after 8 Gy of IR in
HCT116 cells. (D) DNA break measurements under conditions like those
in (C) in HCT116 cells. Data are means ± SEM;N= 3,n> 100. *P< 0.0443
(Kruskal-Wallis multiple-comparisons test). (E) Protein sequence analysis

(Clustal Omega) of ICAD indicates two conserved ATM/ATR SQ phosphoryl-

ation motifs. (F) ICAD is phosphorylated at Ser^107 and Ser^257 after IR.

ICAD KO U2OS cells expressing GFP, WT ICAD, or DSA ICAD were irradiated

(8 Gy) and GFP-tagged proteins enriched by GFP trap. Immunoblotting

with phospho-specific antibodies to Ser^107 and Ser^257 on ICAD. (G) DSA-

ICAD is not stably recruited to laser-microirradiated damage. U2OS cells

expressing GFP-WT ICAD or GFP-DSA ICAD were laser-microirradiated and

imaged every minute for 25 min. Data are means ± SEM; cells per biological

replicate,n= 7; biological replicates,N=3.(H) RPA foci in indicated cells

24 hours after IR;N= 3,n> 50, box-and-whisker plot shows median and

10th to 90th percentiles. *P= 0.0324 [multiple-comparisons analysis of

variance (ANOVA)].

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