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. 2017 Jun 29;37(14):e00659-16. doi: 10.1128/MCB.00659-16

FIG 4.

FIG 4

DNA damage from replication stress in HGPS cells colocalizes with pRPA32 and not with telomeres (TRFI). (A) Immunostaining for γH2AX and pRPA32 in cycling AG11 cells. Phospho-RPA32 (Ser33) serves as a marker for single-stranded DNA that is associated with stalled DNA replication forks. (B) Western blot analysis of phospho-ATR and total ATR in cycling (C) and senescent (S) AG08 and AG11 cells. β-Actin served as a loading control. UV-treated normal cells (AG08) served as a positive control for pATR expression. (C) Immunostaining for γH2AX, TRF1, and pRPA32 in AG11 HGPS cells. pRPA is a marker for single-stranded DNA that accumulates at stalled DNA replication forks, and TRF1 is a marker for telomeres. AG11 cells were doubly stained with TRF1 and γH2AX or pRPA32 and TRF2. (D) Nucleus of an AG11 cell that was triply stained with pRPA (Dylight 649), TRF1 (Alexa Fluor 488), and γH2AX (Cy3).