pH2AX and 53BP1 staining in PyV MT tumors.

<p><b>A</b>). Paraffin embedded PyV MT tumors were stained with pH2AX antibody (green). Nuclei were counter stained with propidium iodide (PI, red); Inset shows high magnification of a cell from a PyV MT/<i>jnk2+/+</i> tumor with arrows pointing at foci. <b>B</b>). Tumor sections were processed as in A) except incubated with 53BP1 primary antibody. Images were captured and pseudo-colored. 53BP1 antibody (red) and DAPI nuclear stain (green). Inset shows high magnification of a cell from the PyV MT/<i>jnk2+/+</i> with arrows pointing at foci. <b>C</b>). Cells with ≥4 nuclear 53BP1 foci were counted as positive cells in each field; a minimum of 5 fields were counted/mouse. Each point represents a tumor average (p = 0.0286).</p

PyV MT/<i>jnk2</i>−/− cellular response is specific to replication and reversed by ATM/ATR inhibitor caffeine.

<p><b>A</b>). PyVMT/<i>jnk2+/+</i> and PyVMT/<i>jnk2−/−</i> cells were treated with doxorubicin at the indicated concentrations for 18 hours and then lysed. Expression of p21^Waf1, p-p53 (Ser15), pH2AX (Ser139), and cleaved caspase 3 were evaluated using western blot analysis. GAPDH was used to compare even loading amongst samples; <b>B</b>). Cells were treated as described in A). except caffeine 2 mM was added as indicated; <b>C</b>). Cells were treated as described in A). except caffeine 2 and 10 mM were added as indicated. Expression of p21^Waf1, p-p53 (Ser 15), and p53 were evaluated using western blot analysis. GAPDH was used to compare even loading amongst samples.</p

Serum treatment of G₁ arrested cells induces cell death in PyV MT/<i>jnk2</i>−/− cells.

<p><b>A</b>). PyVMT/<i>jnk2+/+</i> and PyVMT<i>/jnk2−/−</i> cells were serum starved for 24 hours and then treated with 10% FBS containing medium. Serum stimulated cells were pulsed with BrdU two hours prior to harvesting and then stained with BrdU primary antibody followed by BrdU detection using flow cytometry. BrdU positivity data are presented as percent positive cells in total cell population; <b>B</b>). PyVMT/<i>jnk2+/+</i> and PyVMT<i>/jnk2−/−</i> cells were serum starved for 24 hours and then treated with 10% FBS containing medium. After 24 hours of serum starvation, cells were either cultured in fresh SFM or medium containing 10% FBS and harvested 24 hours later. Cells were evaluated for Annexin positivity using flow cytometry. Data are expressed as percent positive cells of the total population; <b>C</b>). Cells were serum starved as above and then harvested at indicated time points after 10% FBS stimulation to assess expression of various cell cycle associated proteins using western blot analysis with primary antibodies directed towards the indicated proteins. GAPDH was used to compare even sample loading.</p

PyV MT/<i>jnk2</i>−/− cells experience replicative stress and increased p21^Waf1 expression.

<p><b>A</b>). Cells were serum starved and then harvested at different time points after 10% FBS stimulation to evaluate CDT1, p21^Waf1, p-Chk1 (Ser345), and p-p53 (Ser 15) expression by western blot analysis using primary antibodies directed towards the indicated proteins. CDT1 expression at each time point was normalized to GAPDH and graphed for PyVMT/<i>jnk2+/+</i> and PyVMT/<i>jnk2−/−</i> cell lines; <b>B</b>). PyVMT/<i>jnk2+/+</i> and PyVMT/<i>jnk2−/−</i> cell lines were infected with either adenoviral-GFP or adenoviral-CDT1. Forty-eight hours later, cells were stained using PI with RNase, and then evaluated for cell cycle distribution using flow cytometry; <b>C</b>). Cells were infected with either adenoviral-GFP or adenoviral-CDT1 and harvested 24 hours later. Alternatively, cells were treated with hydroxyurea (HU 5 mM) for 24 hours and then harvested. Western blot analysis was used to measure pChk1 (Ser 345) and p21^Waf1 expression. GAPDH was used to compare sample loading; <b>D</b>). Cells were infected with either adenoviral-GFP or adenoviral-CDT1 during 24 hours of serum starvation then stimulated with 10% FBS and harvested 24 hours later. pChk1 (Ser 345), p-p53 (Ser15), and p21^Waf1 expression was evaluated using western blot analysis. GAPDH was used to compare sample loading.</p

Target tumors obtained from PyV MT/<i>jnk2</i>−/− had increased aneuploidy.

<p><b>A</b>). Tumors were finely minced and digested as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010443#s4" target="_blank">Methods</a> section. Cells were trypsinized at passage 2 to 3 and assessed for DNA content using PI staining. PyV MT<i>/jnk2−/−</i> tumors contained more cells with DNA content ≥4N (PyV MT/<i>jnk2+/+</i> (n = 5), PyV MT/<i>jnk2−/−</i> (n = 10), p = 0.0485); <b>B</b>). Primary cells were treated with colcemid and harvested to assess number of chromosomes per metaphase. The number and frequency of abnormal (aneuploid) chromosome numbers are higher in PyV MT<i>/jnk2−/−</i> compared to PyV MT/<i>jnk2+/+</i> (p = 0.0043). Each colored bar represents a single target tumor obtained for an individual mouse. The number to the right of the mouse number denotes the number of metaphases counted for each target tumor. The graph illustrates the frequency of a particular chromosome number for each target tumor; <b>C</b>). Tumor lysates were subjected to SDS PAGE and western blotting using p53 primary antibody and detected using chemiluminescence. GAPDH primary antibody was used to assess similar loading amongst samples.</p

JNK2 is integral in sensing replicative stress and localizing at RPA coated lesions.

<p><b>A</b>). PyVMT/<i>jnk2−/−</i> cells were infected with JNK2<i>α</i> retrovirus and selected using puromycin. GFP-JNK2 expression was measured using JNK2 primary antibody and PyVMT/<i>jnk2+/+</i> lysates as positive a control; <b>B</b>). PyVMT/<i>jnk2−/−</i> and PyVMT/<i>jnk2−/−GFP-JNK2a</i> expressing cells were infected with increasing doses of GFP-CDT1. Cells were processed as described in C). Cell lysates were analyzed for pChk1 (Ser 345), p53 (Ser 15) and p21^Waf1. GAPDH was used to compare even sample loading. <b>C</b>). MCF10A cells were plated in chamber slides, untreated or treated with UV (10 J/m²), and fixed 2 hrs later. Cells were incubated with RPA, DNA Ligase 1 (Lig1), PCNA, or JNK2 primary antibodies, as indicated, followed by incubation with FITC or Texas Red secondary antibodies, (G) Green, (R) Red. Panel D includes images acquired using confocal microscopy. Co-localization was evaluated using color overlay.</p

Genes of interest on chromosomal segments.

<p>Genes of interest on chromosomal segments.</p

Systemic <i>jnk2</i> deletion enhances tumor development.

<p>PyV MT<i>/jnk2+/+</i> (n = 12), PyV MT/<i>jnk2+/−</i> (n = 16), and PyV MT/<i>jnk2−/−</i> (n = 19) mice were palpated for mammary tumors thrice weekly. Once palpated, tumor growth was recorded thrice weekly. <b>A</b>). Kaplan Meier graph showing age of first tumor palpation (median age was day 55 for PyV MT<i>/jnk2−/−</i> vs. day 70 for PyV MT<i>/jnk2+/+</i>, p = .11); <b>B</b>). Total number of tumors palpated per mouse at the time of harvest was higher in PyV MT/<i>jnk2−/−</i> mice compared to PyV MT/<i>jnk2+/+</i> mice, p = 0.0192); <b>C</b>). Paraffin embedded, non-target tumor sections were probed with cleaved caspase 3 primary antibody and detected using FITC labeled secondary antibody. Nuclei were stained with propidium iodide. The total number of cells staining positive for cleaved caspase 3 were scored and divided by the total number of nuclei (n = 5 tumors in each group); <b>D</b>). Paraffin embedded tissue sections were probed with Ki-67 primary antibody and detected using DAB. Cells staining positive for Ki-67 were counted and divided by the total number of nuclei (Hematoxylin) per field. Five fields per tumor were counted (n = 5 per genotype, p = 0.0159); <b>E</b>). Paraffin embedded tissue sections were probed with p-c-Jun (Ser63) primary antibody and detected using DAB. Hematoxylin was used as a nuclear stain.</p