Cell apoptosis in the presence of O-DDHSL.

<p><b>A</b>- Panc-1, Aspc-1 and HPDE-1 cells (6×10³ cells per well) in triplicates was treated with 300 (Panc-1 and HPDE) or 200 µM (Aspc-1) of O-DDHSL, respectively, for 24 h. Cell apoptosis was assayed using a fluorescent based caspase 3/7 activity detection kit. While apoptosis in both Panc-1 cells (P = 0.047*) and Aspc-1 cells (P≤0.02**) were significant, HPDE cell apoptosis was marginally significant (P = 0.059***) <b>B</b> – Light micrographs of Panc-1, Aspc-1 and HPDE cells before and after exposure to O-DDHSL at concentrations of 300 µM (Panc-1 and HPDE) and 200 µM (Aspc-1), respectively, for 48 h (20×). <b>C</b> – Cell proliferation of HPDE, Panc-1 and Aspc-1cells upon exposure to O-DDHSL decreased by 35% (P = 0.042*), 47% (P≤0.035 **), and 52% (P≤0.025***), respectively.</p

Effect of HSL on cell viability.

<p><b>A</b>- Panc-1 (6×10³ cells per well) was treated with different concentrations of O-DDHSL for 24 and 48 h. Significant decrease in cell viability was observed with 200–300 µM O-DDHSL (P≤0.05) after 24 h. At 48 h, cell viability decrease was significant at O-DDHSL concentration at or above 100 µM (P≤0.02; n = 3). However, HPDE cell viability was not affected after 48 h except for a slight decrease at 300 µM O-DDHSL. No effect was observed with O-HHSL. <b>B</b> – Aspc-1 (6×10³ cells per well) was more sensitive to O-DDHSL exposure than Panc-1. A significant decrease (P≤0.02) was observed in viability ≥25 µM O-DDHSL after 24 or 48 h (n = 3). Cell viability was not affected by O-HHSL. In both cases, DMSO (0.02%) was used as diluent control which did not affect the cell viability <i>per se</i>. <b>C–E</b> Panc-1, Aspc-1 and HPDE cells were plated on a layer of matrigel in chamber slides in serum free DMEM/F12 media and allowed to grow for two weeks. O-DDHSL (200 µM) was added to the cells and incubated for 48 h at 37°C. Subsequently, a fluorescent dye Calcein AM was added and further incubated for 2 h for its uptake by the cells. C–E – Light microsopy pictures of cells growing on matrigel before and after addition of O-DDHSL showing morphological changes of apoptosis (top panel). Bottom panel shows the uptake of Calcein AM dye in the cells before and after treatment with O-DDHSL, showing dye uptake by viable cells and loss of cell viability resulting in the absence of dye uptake (40×).</p

Colony formation upon treatment with O-DDHSL.

<p><b>A</b>- Colony forming assay was performed by plating 2×10⁴ cells per plate with or without treatment of Panc-1, Aspc-1 and HPDE cells with O-DDHSL (150 µM) for 48 h. After seven to ten days incubation at 37°C the cells were fixed with 70% methanol and stained with 0.5% crystal violet. Cell colonies were counted using Open CFU software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106480#pone.0106480-Geissmann1" target="_blank">[21]</a>. Colonies, defined as groups of ≥25 cells, were identified and classified as a single colony. <b>B</b> – Relative colony formation depicted as a percentage between O-DDHSL untreated and treated cells, Panc-1(P≤0.029*), Aspc-1(P≤0.03**) and HPDE (P≤0.05***).</p

Cell migration by wound healing assay.

<p><b>A</b>- Panc-1, Aspc-1 and HPDE cells were plated and allowed to grow as a monolayer after which a similar sized scratch was made with a sterile tip in the culture plates. The plates were photographed at baseline (0 h). A set of plates were treated with O-DDHSL (150 µM) and the gap closure was photographed after 48 h. In the presence of O-DDHSL, the migration of cells was considerably less, which resulted in non-closure of the wound gap compared to untreated cells (n = 3). Red arrows - Wound gap (20×). <b>B</b> - The wound area in the image was measured using Image J software and the baseline gap area was assigned an arbitrary number of 1. The untreated carcinoma cell wound closure values at 48 h (P = 0.027*) was significant. The O-DDHSL treated cell wound closure value at 48 h for both Pan-1 and Aspc-1 was not significant (P≥0.05). In HPDE cells, the wound closure between treated and untreated cells was almost similar. <b>C</b> – Detection of a fluorescent analog of O-DDHSL, N-Dd-HSL-3-HF, 10 µM, in different cells after 30 min at 37°C (40×).</p

qRT-PCR analysis of genes <i>Rho C, Cofilin</i> and <i>IQGAP-1</i> in different cells.

<p>In order to analyze the changes in mRNA expression of <i>RhoC</i>, <i>cofilin</i> and <i>IQGAP-1</i> in Panc-1, Aspc-1 and HPDE cells, upon treatment with O-DDHSL (150 µM; 48 h), a qRT-PCR was performed with specific primers. <i>β-actin</i> was used as a housekeeping gene. Basal expression of all the above genes was observed in HPDE cells. <i>Cofilin</i> mRNA level increased in Panc-1 (P = 0.04*) and HPDE cells (P = 0.037*). Similarly, the RhoC mRNA levels increased in Panc-1 (P = 0.032**) and HPDE cells (P = 0.029**), respectively. The <i>IQGAP-1</i> mRNA level decreased in HPDE cells (P = 0.024***). In Aspc-1 cells, no change was observed in <i>IQGAP-1</i> mRNA, but a marginal decrease in <i>cofilin</i> and two fold (P = 0.036**) increase in <i>RhoC</i> mRNA was observed.</p

Chemical structures of homoserinelactones.

<p>Chemical structures of homoserinelactones.</p

Y-chromosome DNA Is Present in the Blood of Female Dogs Suggesting the Presence of Fetal Microchimerism

<div><p>Fetal microchimerism has been suggested to play contradictory roles in women’s health, with factors including age of the recipient, time elapsed since microchimerism occurred, and microchimeric cell type modulating disease. Both beneficial and harmful effects have been identified in wound healing and tissue regeneration, immune mediated disease, and cancer. This area of research is relatively new, and hindered by the time course from occurrence of fetal microchimerism to the multi-factorial development of disease. Dogs represent an excellent model for study of fetal microchimerism, as they share our environment, have a naturally condensed lifespan, and spontaneously develop immune-mediated diseases and cancers similar to their human counterparts. However, fetal microchimerism has not been described in dogs. These experiments sought preliminary evidence that dogs develop fetal microchimerism following pregnancy. We hypothesized that Y chromosomal DNA would be detected in the peripheral blood mononuclear cells of female dogs collected within two months of parturition. We further hypothesized that Y chromosomal DNA would be detected in banked whole blood DNA samples from parous female Golden Retrievers with at least one male puppy in a prior litter. Amplification of DNA extracted from five female Golden Retrievers that had whelped within the two months prior to collection revealed strong positive bands for the Y chromosome. Of banked, parous samples, 36% yielded positive bands for the Y chromosome. This is the first report of persistent Y chromosomal DNA in post-partum female dogs and these results suggest that fetal microchimerism occurs in the canine species. Evaluation of the contributions of fetal microchimeric cells to disease processes in dogs as a model for human disease is warranted.</p></div

Dog Characteristics and Gel Analysis.

<p>Animal number: Corresponds to the gel lanes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068114#pone-0068114-g002" target="_blank">Figure 2</a>; Result: ‘+’ denotes FMC positive; blank denotes negative; Intensity: relative intensity of Y chromosomal band to background gel; Months since parturition: duration of time passed since last litter containing male offspring to date of blood draw; Male sibling if no litters prior: presence of male sibling in birth litter if nulliparous at time of blood draw.</p

PCR reactions for positive and negative controls.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068114#pone-0068114-g001" target="_blank">Figure 1A</a> represents the native primer for dog Y-specific DNA fragment of 650 bp, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068114#pone-0068114-g001" target="_blank">Figure 1B</a> represents the nested primer or ∼320 bp within the 650 bp fragment.</p

Nested PCR of dilutions of male: female blood, with male to female ratios of 1∶1 to 1∶90,000.

<p>Bands of Y chromosomal DNA are detected in as small as 1∶60,000-fold M:F diluted samples, but not in 1∶90,000-fold diluted samples.</p