Chromosome Cycle of Meiosis and Sexual Reproduction

<div><p>(A) Simplified reproductive cycle of sexual organisms. Chromosomes are represented as colored lines, with two of each color to represent the maternal and paternal copies of each. During meiosis, the number of chromosomes is reduced by half to generate gametes (e.g., sperm and eggs). Diploidy is restored upon fertilization.</p> <p>(B) Two consecutive cell divisions in meiosis. In meiosis I, homologous chromosomes recombine and exchange genetic information before they separate from one another. In meiosis II, sister chromatids separate.</p></div

Meiotic Recombination Pathway as Elucidated in S. cerevisiae

<p>Recombination is initiated by Spo11-generated DSBs. (The DNA duplex from only one of two sister chromatids is shown.) Spo11 is then released and the 5′ ends of DSBs are degraded to yield 3′ single-stranded DNA tails. The single-stranded DNA invades one of the two sister chromatids of the homologous chromosome (only one of the homolog's chromatids is shown). Strand invasion is catalyzed by Dmc1 (and/or Rad51, not shown). Completion of repair yields either reciprocal exchange of chromosome arms that flank the break (a crossover) or no exchange of flanking arms (a noncrossover). In <i>rad50S</i>-like mutants, Spo11 remains covalently attached to DNA, and the DSBs are not repaired. In <i>dmc1</i> mutants, DSBs persist with long, single-stranded DNA tails.</p

Recombination Frequency Mapped at Different Resolutions

<p>The upper plot is a highly schematic representation of recombination distribution along an entire chromosome, showing alternating domains of relatively higher (“hot”) and relatively lower (“cold”) recombination activity. Such domains are megabase pairs long in mammals and 20–100 kb long in yeast. When examined at higher resolution, distinct hot spots are revealed, separated by DNA segments where little, if any, recombination occurs (lower plot). Recombination hot spots in mammals are typically ∼1–2 kb wide, separated by tens of kilobases of recombinationally suppressed DNA. For examples of real recombination distributions, see [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050333#pbio-0050333-b006" target="_blank">6</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050333#pbio-0050333-b025" target="_blank">25</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050333#pbio-0050333-b026" target="_blank">26</a>].</p

miR-192 directly targets IL-17Rs, including IL-17RA and IL-17RE.

<p>(A) The predicted miR-192 and miR-215 binding sites within 3′-UTRs of IL-17RA, IL-17RC and IL-17RE, and their mutated versions by site mutagenesis. Upregulation of miR-192 and miR-215 (B) repressed expressions of IL-17RA, IL-17RC and IL-17RE at both protein (C) and mRNA (D) level. (D)The repression of luciferase activities by 3′-UTRs of IL-17RA and IL-17RE, but not IL-17RC, was dependent on miR-192 and miR-215. Mutated 3′-UTRs of IL-17RA and IL-17RE abrogated miR mediated repression of luciferase activity. (**P<0.01, Figure is representative of 3 experiments with similar results.).</p

The IL-17/miR-192/IL-17Rs feedback loop is characteristic for MM.

<p>Correlative analysis of the indicated mRNAs and miR-192 in bone marrow tissues of patients with MM was detected by qPCR. Spearman correlation coefficient with the respective significance was indicated.</p

IL-17 directly repressed expression of miR-192.

<p>(A) MM1S cells were treated with IL-17 for 12 h and miR-192 expression was determined by qPCR analysis. IL-17 treatment significantly downregulated miR-192 in a dose-dependent manner. (B) IL-17 treatment significantly downregulated miR-192 in MM1R cells and H929 cells. (C) IL-17 treatment activated p65 pathway. (D) Location and sequence of predicted p65-binding sites in the promoter of miR-192 gene. (E) ChIP assay was performed and indicated that p65 could bind to the indicated regions of miR-192 promoter. (F) siRNA-mediated downregulation of p65 prevented the repression of miR-192 after IL-17 treatment. (**P<0.01, *P<0.05, Figure is representative of 3 experiments with similar results.).</p

Lung tumor spheres have high tumorigenic potential.

<p>Tumorigenic potential of tumor sphere cells is greater than monolayer cancer cells. Monolayer and tumor sphere cells from A549 were injected s.c. into athymic nude mice at concentrations of 1×10⁴ cells/ml. Mice were sacrificed at day 60, and tumor was surgically removed and measured, and the mean values of the tumor size were shown in the table.</p

Effects of IL-17 on proliferation, apoptosis, adhesion, migration and EMT properties of MM1S cells.

<p>(A) IL-17 induced cell proliferation was analyzed by WST-8 assays (left) and Trypan Blue exclusion viable cell assay (right). (B) IL-17 inhibited cell apoptosis determined by Annexin-V binding assay (top), Ki-67 staining (middle), and TUNEL assay (bottom). (C) Canonical histogram of apoptotic rate characterized by Annexin-FITC positive cells was shown. (D) IL-17 decreased cell adhesion to fibronectin and collagen I. (E) IL-17 increased cell migration. (F) IL-17 induced EMT and Rac1 expression of cells. Epithelial marker E-cadherin, mesenchymal marker Vimentin, EMT transcription factors Snail and Slug, and Rac1 expression were detected by western blot analysis. Note all the effects induced by IL-17 were in a dose-dependent manner. (**P<0.01, *P<0.05, Figure is representative of 3 experiments with similar results.).</p

Chemoresistence of monolayer and tumor sphere.

<p><b>A.</b> cisplatin, <b>B.</b> doxorubicin resistance of monolayer culture and tumor spheres were analyzed. Cells dissociated from lung tumor spheres or monolayers were plated out in 96-well plate at 2×10⁴ cells/ml, 24 h later, media containing different concentrations of cisplatin or doxorubicin was added. After 48 h culturing, 10% Alamar Blue was added and read at 544/590 nm, the percent viability was calculated relative to cells not exposed to any chemotherapeutic drugs. The results represent the means ± SD.</p

SP and NSP cells form tumor spheres with equal capacity in human adenocarcinoma cell lines.

<p><b>A.</b> Characteristic Hoechst 33342 dye staining profile demonstrating the existence of SP (polygon gated) and NSP (ellipse gated) cells in human A549 and H1299 adenocarcinoma cells. Percentage of SP cells is indicated. Cells were stained with 5 mg/ml Hoechst33342 (HO), and control cells were also co-stained with 10 mM fumitremorgin C (FTC) to specify the SP cells. Cells were sorted using a Beckman MoFlo cytometer and data were annotated using FCS Express. <b>B.</b> Phase-contrast photographs of lung tumor spheres obtained from SP and NSP cells. SP and NSP cells (1000 cell/ml) were plated onto ultra low adherent flasks in serum free media supplemented with growth factors and cultivated as described in Material and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013298#s2" target="_blank">Methods</a>. <b>C.</b> Re-stain of SP and NSP cells from A549 and H1299 with Hoechst 33342 dye. Percentage of SP cells is labeled.</p