Ectopic K164R-mutated PCNA forms hetero-trimers with endogenous PCNA, depletes endogenous PCNA homo-trimers, and disturbs DDT.

<p>(A) Immunoprecipitation assays showing the formation of hetero-trimers of exogenous PCNA[KR] and endogenous PCNA in WI38VA13 cell transformants. The chromatin fractions (20% input) prepared from vector-transfected cells (v) and cells expressing varying levels of HA-PCNA[KR] (clone #1 = #2 > #3) were immunoprecipitated using an anti-HA antibody and then analyzed by immunoblotting using an anti-PCNA, anti-HA, or anti-Lamin B (control) antibody. The whole cell lysates (WCLs) and 20% unbound samples were also analyzed. The ratios of the signal intensities of endogenous PCNA detected in the unbound fractions of clones #1, #2, and #3 to control cells (v) are indicated below the upper panel. (B) Cell-cycle progression analyses of vector-transfected WI38VA13 cells and the HA-PCNA[KR] clone #1, #2 and #3 cells. The cells were synchronized by the double thymidine block method, mock-irradiated (UV-) or irradiated with 8 J/m² UVC (UV+) at the released time point, and then incubated for the indicated periods. The cell-cycle profiles were obtained by FACS analysis. The percentages of cells in the S-phase at the 15 h time point are indicated.</p

Forced increase of mono-ubiquitinated PCNA does not restore the DDT defects of PCNA[KR] cells.

<p>PCNA-wild-type (WT) and PCNA[KR] cells were transfected with empty vector (v) or FLAG-Polη (η) or FLAG-RAD18 (18) expression constructs. (A) Cells were mock-irradiated (UV-) or irradiated with 15 J/m² UVC (UV+), incubated for 3 h, and then subjected to immunoblotting using an anti-PCNA, anti-Polη, anti-RAD18, and anti-Lamin B antibodies. (B) Cells were co-transfected with GFP and empty vector or the FLAG-Polη or FLAG-RAD18 expression construct, and then treated with 2.5 mM thymidine for 24 h to concentrate the G1/S-phase populations. The cells were then mock-irradiated (UV-) or irradiated with 8 J/m² UV and cultured for the indicated periods. The cell-cycle profiles of the GFP-positive populations were determined by FACS analysis. The percentages of cells in the S-phase populations at the 12 h time point are indicated. (C) Cells were co-transfected with His-Ub and empty vector (v) or the FLAG-Polη (η) expression construct, mock-irradiated (-) or irradiated with 15 J/m² UVC (+), and then incubated for 3 h. Ni-pull-down assays were performed using the chromatin fractions (10% input) and samples were analyzed by immunoblotting using an anti-PCNA or anti-Polη antibody.</p

PCNA[KR] cells are sensitive to DNA-damaging agents.

<p>The sensitivities of WI38VA13-derived cells to UV irradiation (A) or cisplatin (Cis-Pt) (B) with and without siRNA-mediated knockdown of endogenous PCNA (siPCNA). Clonogenic survival rates were determined after UVC irradiation or cisplatin treatment (24 h) of the indicated cell strains. Data are represented as the mean ± standard deviation of n = 3 independent experiments.</p

PCNA[KR] cells show DNA replication problems after UV irradiation.

<p>(A, B) S-phase progression analysis. Vector-transfected, PCNA-wild-type (WT) or PCNA[KR] WI38VA13 cells were synchronized using the double thymidine block method, mock-irradiated (UV-) or irradiated with UVC at the released time point, and then incubated for the indicated periods prior to FACS analysis of the cell-cycle profile. (C) Immunoblot analysis of CHK1 phosphorylation. Cells were irradiated with 10 J/m² UVC and incubated for the indicated periods. Immunoblotting was performed using anti-phospho-CHK1 (Ser345), anti-CHK1 and anti-actin antibodies. (D) Analysis of γH2AX-positive populations. The indicated cells were irradiated with 5 J/m² UVC and incubated for the indicated periods. The cells were then fixed and stained with an anti-γH2AX antibody (shown in red). Nuclei were visualized by Hoechst 33342 staining (shown in blue).</p

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.</p

Ectopic expression of K164R-mutated PCNA disturbs multiple mono-ubiquitinations of PCNA trimers in human cells.

<p>(A) Immunoblot analyses of endogenous and ectopically expressed PCNA in vector-transfected, PCNA-wild-type (WT)-expressing and PCNA[KR]-expressing WI38VA13 cells that were untreated (untreat) or transfected with a nontargeting control (NTC) or PCNA-specific (siPCNA) siRNA. Four days after transfection, the cells were mock-irradiated (UV-) or irradiated with UVC at 10 J/m² (UV+), and then incubated for a further 3 h. Lamin B was detected as a loading control. (B) Ni-pull-down assay of PCNA-WT and PCNA[KR] cells transiently transfected with empty vector (vec) or the His-Ub expression construct (His-Ub). The cells were incubated for 36 h, mock-irradiated (-) or irradiated with 15 J/m² UVC (+), and then incubated for a further 3 h. Ni-pull-down assays were performed using the chromatin fractions (5% input) and samples were analyzed by immunoblotting using an anti-PCNA or anti-Ub antibody. WCL, whole cell lysate.</p

PCNA homo-trimers undergo multiple-mono-ubiquitinations <i>in vivo</i>.

<p>The results of a pull-down assay using stably-expressed His-Ub. WI38VA13 cells stably expressing His-Ub (His-Ub) or vector-transfected control (vec) were mock-irradiated (-) or irradiated with 15 J/m² UVC (+) and incubated for 3 h. Ni-pull-down assays were performed using the chromatin fractions (2% input) and samples were analyzed by immunoblotting using an anti-PCNA or anti-Ub antibody. WCL, whole cell lysate.</p

The ATM/TP53/p21 pathway maintains genomic integrity under chronic γ-irradiation conditions by regulating cell-fate decisions.

<p>(A) BJ1/hT cells were transfected with indicated siRNAs and cultured for 4 days under chronic γ-irradiation conditions (0.694 mGy mGy/min). β-gal activity was used to assess cellular senescence. Values represent the mean ± SD of three independent experiments. ***<i>P<0.001</i>. (B) BJ1/hT cells were transfected with indicated siRNAs and then cultured under chronic γ-irradiation conditions at indicated dose rates for 4 days. After an additional 10 days of growth in unirradiated conditions, the number of macroscopic colonies was determined. Values were normalized to control and represent the mean ± SD of three independent experiments. (C) BJ1/hT cells were transfected with indicated siRNAs and then cultured under chronic γ-irradiation conditions at indicated dose rates for 4 days (-d4). Some cells were cultured an additional 4 days under unirradiated conditions (-d4-d8). Values represent the mean ± SD of three independent wells. (D) Wild-type or <i>TP53</i> null mice were exposed to acute (ii, iv, vi) or chronic (iii, v, vii) γ-irradiation treatment conditions, as indicated on the left. β-gal activity was used to assess cellular senescence of primary lung cells isolated from these mice. Values represent the mean ± SD of independent cultures from 2 mice. *<i>P<0.05</i>. (E) BJ1/hT cells were transfected with indicated siRNA and then cultured without (left) or with (right) KU55933 (10 µM) under chronic γ-radiation conditions at indicated dose rates for 4 days. The number of micronuclei was then assessed. Values represent the mean ± SD of at least three independent experiments.</p

<b>Comparison of colony forming abilities of different types of cells for acute versus chronic γ-irradiation (5 Gy).</b>

<p>Values represent the mean ± SD of three independent experiments.</p>1<p>Survival fractions were determined based on crystal violet staining.</p>2<p>Dose rate of 1.0 Gy/min for 5 min, total dose; 5 Gy. Cells were cultured for 10 days following acute γ-irradiation.</p>3<p>Cells were cultured for 10 days at dose rate of 0.347 mGy/min, total dose; 5 Gy.</p

The dose rate of chronic γ-irradiation affects cell-fate decisions.

<p><b>(A) Colony-forming ability of fibroblasts following chronic γ-irradiation.</b> The experimental scheme is illustrated to the left. TIG-3 p27 cells (2×10²) were exposed to different dose-rates of γ-irradiation for 10 days and then allowed to grow under unirradiated conditions for an additional 10 days. Representative pictures of crystal violet-stained colonies are shown for each dose rate. (B) Cellular senescence induced by acute or chronic γ-irradiation. BJ1/hT cells (4×10³) were exposed to the treatment condition indicated on the left. Grey arrows indicate chronic γ-irradiation at indicated dose rates. Black arrows indicate acute γ-irradiation of indicated doses. Following 1 day of recovery and 6 days of additional growth, cells were stained for β-gal activity to assess levels of senescence (right). Values represent the mean ± SD of three independent experiments. *<i>P<0.05</i>, ***<i>P<0.001</i>. (C) Cellular senescence induced by acute or chronic γ-irradiation <i>in vivo</i>. Female C57BL/6 mice were exposed to the treatment condition indicated to the left. Grey arrows indicate chronic γ-irradiation at indicated dose rates. Black arrows indicate acute γ-irradiation of indicated doses. Primary cells isolated from lungs of irradiated mice were stained for β-gal activity to assess levels of senescence (right). Values represent the mean ± SD of independent cultures from 2 mice. ***<i>P<0.001</i>.</p