Polymorphisms in APOBEC 3 Genes.

<p>(A) Different splice variants and polymorphic exons found in different inbred mouse strains. Blue boxes denote exons found in inbred strains such as BALB/c and 129; red boxes denote exons with polymorphic amino acid differences in C57BL-derived strains. The table denotes the relative expression levels of the different splice variants expressed in the different strains, as well as the susceptibility (S) or resistance (R) to F-MLV infection. Data taken from references <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000347#ppat.1000347-Santiago1" target="_blank">[7]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000347#ppat.1000347-Takeda1" target="_blank">[8]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000347#ppat.1000347-Miyazawa1" target="_blank">[65]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000347#ppat.1000347-Okeoma2" target="_blank">[66]</a>. (B) Different <i>A3</i> alleles found in human populations. At least seven SNP polymorphisms have been found in the A3G genes in humans, although only one polymorphic <i>A3G</i> allele, H186R, has been associated with increased susceptibility to HIV-1-mediated disease <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000347#ppat.1000347-An1" target="_blank">[69]</a>. Also shown is the presumed recombination event leading to the deletion of part of A3B in some individuals and a fused h<i>A3A</i>/<i>3B</i> gene <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000347#ppat.1000347-Kidd1" target="_blank">[71]</a>.</p

NMuMG cells transduced with <i>Tcf7l2</i> and <i>Antxr1</i> show enhanced response to an exogenous WNT signal.

<p>A) NMuMG cells stably transduced with the CIS genes were grown in control (Control CM) or WNT3A (WNT3A CM) conditioned media for 48 hr. The level of Axin2 was then examined by RT-qPCR. All values were normalized to GAPDH. Data are the means of three independent experiments with the SE values indicated by error bars. **p≤0.01, as determined by Student's t-test. B) <i>Tcf7l2</i> and Antxr1-expressing cells were incubated with increasing dose of WNT3A CM for 48 hr (1, 10, 100, 1000 · l in 10 ml media) and <i>Axin2</i> RNA levels were examined by RT-PCR. GAPDH was used as internal control.</p

Integration sites in tumors with novel CIS.

<p>All of the identified integration sites in tumors with novel CIS (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027425#pone-0027425-t001" target="_blank">Table I</a>) are listed. Additional CIS found in the tumors are underlined.</p

Expression of selected CIS genes in MMTV-induced mammary tumors.

<p>RNA was extracted from eight MMTV-induced mammary tumors; 5 from C3H mice (BF, BN, C, F, P) and 2 from BALB/c mice (5, 11). Normal mammary gland (MG) was obtained from a 4 month old virgin mouse and used as a control. Expression of selected CIS genes (<i>Wnt1</i>, <i>Prkaca</i>, <i>Arhgap18</i>, <i>Tcf7l2</i>, <i>Antxr1</i>) were measured by qRT-PCR. GAPDH was used as the endogenous control gene. The relative levels of the gene specific PCR product were normalized to GAPDH. All error bars represent standard deviations. Arrows indicate tumors harboring the corresponding CIS.</p

Basal WNT signaling pathway in the CIS-transduced cells.

<p>A) Untransduced NMuMG cells were incubated with 20% Wnt3a- or Wnt5a-containing media for 48 hr. Nuclear protein was extracted and active β-catenin levels were analyzed by immunobloting. Samples were also probed with the anti-lamin B antibody to verify equal loading. B) Nuclear extracts were obtained from NMuMG cells transduced with CIS genes and active β-catenin level was measured by immunobloting. Samples were also probed with the anti-lamin B antibody to verify equal loading. C) mRNA was extracted from NMuMG cells transduced with CIS genes and RT-qPCR was used to measure Ccnd1, Myc, and Tfr1 RNA levels. All values were normalized to GAPDH. Data are the means of three independent experiments with the SE values indicated by error bars. Data were analyzed by Student's t-test. *p≤0.05; **p≤0.01 versus the corresponding control value, as determined by Student's t-test.</p

Increased expression of CIS genes causes morphological transformation of mammary epithelial cells in 3D culture.

<p>A) NMuMG or HC11 cells transduced with the different CIS genes or empty vector (control) were grown in 8 well chamber slides with matrigel. Images were captured at day 10 (200X magnification). B) Acinar size was measured on day 10 of growth in matrigel. Acini with diameters over 50 µm were scored (n = 30 for each cell line). **p≤0.01, determined by Student's t-test.</p

Expression of target CIS proteins in transduced mammary epithelial cells and enhanced cell growth.

<p>The pBabe retroviral constructs containing the cDNAs of <i>Wnt1</i>, <i>Arhgap18</i>, <i>Tcf7l2</i>, and <i>Antxr1</i> were transduced into NMuMG and HC11 mouse mammary cells. A) Cell extracts from the different transduced cells were subjected to western blot analysis using anti-HA antibodies. B) Cells stably transduced with CIS genes were plated in 96-well plates and grown for 4 days. Cell growth was monitored by the MTT assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027425#s4" target="_blank">Materials and Methods</a>). Data represents mean values with the SE (n = 5). Data were analyzed by two way ANOVA analysis with Dunnett's post hoc test. At day 4, all CIS-transduced NMuMG cells and all but the <i>Tcf7l2</i>-transduced HC11 cells showed significant differences from control cells (p≤0.01).</p

Expression of CIS genes in transduced mammary epithelial cells enhances cell proliferation.

<p>A) FACS plots for different transduced cells. S, percentage of cells in S phase averaged from 3 independent samples. *p≤0.05, determined by Student's t test. B) Bar graphs show the percentage of cells in G0/G1 (shaded), M (open) and S (stippled phase). Shown is the average of 3 experiments; error bars show the SE.</p

Selected CISs found in MMTV-induced mammary tumors.

<p>*Distance in kb from start of transcription for CIS other than <i>Wnt</i> or <i>Fgf</i> genes.</p><p>**expression in normal mammary tissue (data from BioGPS).</p>+<p>number of BALB and C3H tumors with this CIS.</p>#<p>data from Oncomine database. Abbreviations: HBC, human breast cancer; LBC, lobular breast carcinoma; IBC, invasive breast carcinoma; ILC, invasive lobular carcinoma; DBC ductal carcinoma.</p

RT-PCR analysis of BALB/c (B) and C3H (C) tumors.

<p>CIS: <i>Wnt</i> or <i>Fgf</i> integration identified by cloning. Expression: +, expression of oncogene; -, no expression of oncogene; ND, not done.</p