The DDT pathway disturbed by expression of PCNA[KR] is distinct from Polη- and Rev1-mediated TLS.

<p>(A) Immunoblot analyses of PCNA expression in Polη-deficient XP2SASV3 cells stably expressing empty vector (vec), HA-PCNA-wild-type (WT) or HA-PCNA[KR] (KR). The cells were mock-irradiated (UV-) or irradiated with UVC at 10 J/m² (UV+) and then incubated for 3 h. Whole cell lysates were analyzed by immunoblotting using an anti-PCNA or anti-Lamin B (control) antibody. (B) The UVC sensitivities of Polη-deficient XP2SASV3-derived cells (XP-V) expressing empty vector, HA-PCNA-WT or HA-PCNA[KR] after treatment with an siRNA to knockdown endogenous PCNA (siPCNA) or a nontargeting control siRNA (NTC). Data are represented as the mean ± standard deviation (SD) of n = 3 independent experiments. (C) GFP-Polη was transiently expressed in WI38VA13/PCNA-WT or WI38VA13/PCNA[KR] cells treated with an siPCNA or a NTC. The cells were irradiated with 15 J/m² UVC and then incubated for 3 h. After extraction of Triton-soluble materials and fixation, PCNA was detected using an anti-PCNA antibody and nuclei were visualized by Hoechst 33342 staining. A typical nucleus of each cell type is shown. (D) Immunoblot analyses of REV1 and Lamin B (control) expression in WI38VA13 cells expressing empty vector (vec), PCNA-WT (WT) or PCNA[KR] (KR), and transfected with or without a REV1-specific siRNA. Four days after transfection, whole cell lysates were analyzed by immunoblotting using an anti-REV1 or anti-Lamin B antibody. (E) The effects of knockdown of REV1 on the UV sensitivities of the cells described in (D). The ratios of surviving cells exposed to 8 J/m² of UVC irradiation to those that were mock-irradiated were determined. The survival rates of the cells that were not treated with the REV1-specific siRNA (untreat) were taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118775#pone.0118775.g004" target="_blank">Fig. 4A</a>. Data are represented as the mean ± SD of n = 3 independent experiments. (F) Schematic illustration of PCNA mono-ubiquitination-mediated DDT pathways in human cells.