Skip to main content
. 2015 Sep 29;44(1):198–209. doi: 10.1093/nar/gkv957

Figure 2.

Figure 2.

Replication-associated activation of ATM by FA. (A) FA-induced phospho-ATM and phospho-CHK2 localized to EdU-incorporating cells. H460 were treated for 3 h with 300 μM FA. (B) Absence of phospho-ATM and phospho-CHK2 immunostaining in H460 cells with shRNA-depleted ATM. (C) S-phase specificity of FA-induced phospho-CHK2 in H460 and (D) IMR90 cells (300 μM FA, 3 h). Labels: p-CHK2—percentage of cells displaying immunostaining for p-CHK2, p-CHK2/EdU+—percentage of cells that showed both p-CHK2 and EdU staining. Data are Means ± SD for three experiments with each scoring >100 cells. (E) Lack of ATM and CHK2 phosphorylation by FA in growth-arrested IMR90 cells. Data for cycling and confluent cells are from the same blots after removal of intervening lanes. (F) Inhibition of FA-induced ATM signaling in IMR90 cells by aphidicolin (2 μM, added 60 min before FA exposure for 1.5 h). (G) Independence of ATM signaling on the ATR kinase in IMR90 cells. ATRi1 (3 mM caffeine) was added for 1 h before FA exposure for 3 h. (H) Normal ATM and KAP1 phosphorylation in H460 cells in the presence of the ATR inhibitor VE821 (ATRi2, 10 μM). Cells were preincubated for 1 h with ATRi2 and then treated with FA for 3 h. (I) ATM phosphorylation in IMR90 and Seckel syndrome fibroblasts treated for 3 h with FA. (J) ATR inhibitors did not restore ATM activation by FA in the presence of aphidicolin (ATRi1 - 3 mM caffeine, ATRi2 - 10 μM VE821, ATRi3 - 0.5 μM AZ20). Cells were preincubated with 2 μM aphidicolin and ATR inhibitors for 1 h before FA exposure for 2 h.