Figure 2.
PrimPol Is Required for Tolerance of UV Photoproducts in a Pathway Independent of Pol η
(A) Human (HEK293) cells stably expressing PrimPol with a C-terminal Flag-Strep-II tag (PrimPolFlagStrep) were either mock, UV-C (30 J/m2), or X-ray (5 Gy) irradiated or treated for 6 hr with hydroxyurea (HU; 10 nM); following recovery (1 hr for UV-C, 30 min for X-ray, immediately after HU treatment), cells were detergent extracted (0.5% Triton X-100) prior to immunofluorescent (IF) analysis with an anti-PrimPol antibody and DAPI counterstaining.
(B) Representative images of nuclei containing detergent-resistant PrimPol foci.
(C) Proportion of cells in which PrimPol assembled into foci was determined at varying UV-C doses following an 8 hr recovery; error bars indicate SD of three experiments, > 200 cells counted for each dose.
(D) Mock or UV-C irradiated (30 J/m2) cells were allowed to recover for 8 hr before the Triton X-100 (0.5%) insoluble material was collected by centrifugation and treated with DNase and further centrifugated; the resulting samples were analyzed by western blot with anti-PrimPol and PCNA antibodies.
(E) Normal human (MRC5) fibroblasts were either mock (−) or UV-C (30 J/m2) irradiated and, following recovery, were separated into Triton X-100 (0.5%) soluble and insoluble material and analyzed by western blot along with whole-cell extract (WCE).
(F) Normal (MRC5) fibroblasts or XP-V (XP30RO) patient cells were either mock or PrimPol siRNA treated and mock (−) or UV-C (2 J/m2) irradiated and allowed to recover before cell lysates were prepared and analyzed by western blot to determine levels of phosphorylated Chk1 on Ser345.
(G) UV-C clonogenic survival assays were performed with MRC5 and XP30RO cells either mock or PrimPol siRNA treated. Error bars denote SD of three experiments.