SAMHD1 effects on cell proliferation and cell viability in response to DNA damage agents. (A) HeLa cells stably expressing an inducible empty vector (Mock) or a vector encoding for SAMHD1 wild-type (WT) SAMHD1 mutated in the HD domain (HD/AA) or truncated at amino acid 575 (ΔCter) were induced with doxycycline. Results are expressed as the proliferation ratio of SAMHD1-transduced cells divided by mock-transduced cell. A representative experiment out of 3 is shown. (B) HeLa and HeLa cells stably expressing SAMHD1 were treated with increasing concentrations of DNA damaging agents, and the percentage of living cells was measured 3 days after treatment. Each graph shows the mean of 3 independent experiments. *A t-test P < .05. (C) HeLa cells stably expressing SAMHD1 were mock treated or treated with 5 nM of etoposide for 4 or 18 hours. Nuclear extracts were prepared and separated on a glycerol gradient. The localization of SAMHD1 along the gradient was assessed by western blot. (D) HeLa (Mock) and HeLa cells stably expressing Flag-HA–tagged SAMHD1 were treated with 5 nM etoposide for the indicated time. Flag IPs were performed and SAMHD1 interactions with CycA and DCAF1 were assessed by western blot. (E) Flag IPs were performed using nuclear extract of HeLa cells stably expressing Flag-HA–tagged SAMHD1 after treatment with 5 nM etoposide. SAMHD1 and SAMHD1 phosphorylated on threonine 592 (pSAMHD1) levels were determined by western blot. (F) SAMHD1 colocalizes with 53BP1 at the site of DSBs. HeLa cells expressing SAMHD1-HA were mock-treated or treated with 25 nM CPT. SAMHD1 and 53BP1 intranuclear localizations were determined by immunofluorescence using specific antibodies and confocal microscopy.