Descriptive data from studied CD-1 pregnant mice according to treatments groups (n = 5 animals/group): Saline, Cont-Oligo, T-oligo and T-oligo co-treatment with SB203580.

<p>(g: grams; SB203580: p38MAPK inhibitor)</p><p>*Anova, Tukey's Multiple Comparison Test, p = 0.001.</p><p>Descriptive data from studied CD-1 pregnant mice according to treatments groups (n = 5 animals/group): Saline, Cont-Oligo, T-oligo and T-oligo co-treatment with SB203580.</p

Animal models of T-oligo induced senescence.

<p>(A-D) Representative image of 3-nitrotyrosine (3-NT) modified proteins, an oxidative stress marker, in murine fetal membranes. Intrauterine injection of pregnant CD-1 mice were performed with either: A. Saline, B. Cont-oligo, C. T-oligo and D. T-oligo+SB23580 (p38MAPK inhibitor). <b>(</b>E-G) Representative image of Western blot analysis and densitometric quantitation of p38MAPK activation in murine amniotic sac. E. Top panel = phosphorylated (P)-p38MAPK, middle panel = total p38MAPK and bottom panel = β-actin in Cont-oligo, saline, T-oligo+SB203580 (p38MAPK inhibitor) and T-oligo treated mice, respectively. F. Densitometric quantitation P-p38MAPK normalized to total p38MAPK in amniotic sac tissue or G. normalized to β-actin. T-oligo treatment produced a significant (*) increase in P-p38MAPK compared to saline and Cont-oligo treated groups. The co-treatment of T-oligo and SB203580 showed similar results to controls (saline and Cont-oligo). (*ANOVA, p<0.05). Results are representative from 3 animals/ group. (Telomere mimetic overhang sequence (T-oligo, [TTAGGG]₂); control oligonucleotides (Cont-oligo, [AATCCC]₂).</p

DNA damage foci and Toll like receptor (TLR)-9 expression in human amnion cells.

<p>(A) Immunofluorescence staining of phosphorylated (γ) H2AX, a marker for DNA damage response activation. Top panel = T-oligo treated amnion cells, bottom panel = untreated cells. Left panel = γH2AX. Right panel = merged images with DAPI nuclear stain. The bright nuclear dots represent DNA damage foci and are more pronounced in cells treated with T-oligo. (B) Relative quantification of TLR-9 mRNA expression in amnion cells in the studied groups, untreated cells, Cont-oligo and T-oligo treated cells, respectively. Box plots represent the quantification relative to endogenous 16S RNA. Kruskal-Wallis test, p>0.05.(control oligonucleotides (Cont-oligo, [AATCCC]₂); Telomere mimetic overhang sequence (T-oligo, [TTAGGG]₂).</p

Senescence and inflammation induced by T-oligos in CD-1 pregnant mice.

<p>(A-D) Senescence associated β-galactosidase (SA-β-gal) staining of murine amniotic sac. Single blue stained cells indicate β-gal activity. A. saline, B. Cont-oligo, C. T-oligo and D. T-oligo+SB203580 (p38MAPK inhibitor) treated mice. SA-β-gal staining is pronounced in T-oligo treated mice. (E) Concentration of interleukin (IL)-8 protein in murine amniotic fluid. Higher levels of IL-8 were found in T-oligo treated animals compared to controls (saline and Cont-oligo). The production of IL-8 was inhibited by simultaneous treatment with SB203580. (*ANOVA, p<0.05). Results are representative from 3 animals/ group. (Telomere mimetic overhang sequence (T-oligo, [TTAGGG]₂); control oligonucleotides (Cont-oligo, [AATCCC]₂).</p

Quantitation of telomere fragments in human amniotic fluid.

<p>Scatter plot representing the number of telomere fragments detected in amniotic fluid from normal term not in labor (NIL) and term in labor samples. The distribution of telomere fragments significantly differs between groups (p = 0.04; t-test).</p

Human amniotic cells primary cultures.

<p>(A) Immunofluorescent staining of cytokeratin positive amnion epithelial cells. Inset, <b>a.</b> cytokeratin positive cells and <b>b.</b> nuclear staining DAPI. Original magnification x40. (B-D) Cell viability. Representative fluorescence photomicrographs of merged propidium iodide and Hoechst 33342-stained amnion cells. <b>B.</b> Untreated cells, <b>C.</b> Cont-oligo treated cells and <b>D.</b> T-oligo treated cells. Original magnification x40. (E-G) Representative image of Western blot analysis and densitometric quantitation of p38MAPK activation in amnion cells. E. Top panel = phosphorylated (P)-p38MAPK, middle panel = total p38MAPK and bottom panel = β-actin in untreated, Cont-oligo and T-oligo treated cells, respectively. F. Quantitation of P-p38MAPK densitometry normalized to total p38MAPK. T-oligo treatment produced significant (*) increase in P-p38MAPK compared to both untreated and Cont-oligo treated cells. G. Densitometric quantitation of P-p38MAPK normalized to β-actin. Post hoc tests indicated that T-oligo treatment produced significant (*) increase in P-p38MAPK compared to untreated control, but was not significant compared to Cont-oligo treatment. (*ANOVA, p<0.05). (H) Representative image of Western blot analysis of p53 activation in human amnion cells. Top panel = P-p53, middle panel = total p53 and bottom panel = β-actin in untreated and etoposide treated amnion cells, respectively. (I-M) Senescence associated β-galactosidase (SA-β-gal) staining of amnion cells. Single blue stained cells indicate positive β-gal activity. I. Untreated cells, J. Cont-oligo treated cells, K. T-oligo treated cells and L. T-oligo+SB203580 (p38MAPK inhibitor) treated cells. M. Quantification of the positive SA-β-gal cells. Bar graphs represent the differences in the percentage of SA-β-gal staining cells in each group. T-oligo treatment produced a significant increase (*) in the number of senescing cells, which was inhibited by SB203580 treatment. (*ANOVA, p<0.001). (N-Q) Senescence associated sterile inflammation in amnion cells. N. Relative quantification of IL-6 mRNA (p>0.05), <b>O.</b> Relative quantification of IL-8 mRNA in amnion cells (*p = 0.02), P. Protein concentration of IL-6 in conditioned media (*p<0.0001), and Q. Protein concentration of IL-8 in conditioned media (*p = 0.001). The production of IL-8 and IL-6 was inhibited by simultaneous treatment with SB203580. * ANOVA, T-oligo treated samples significantly higher compared with untreated, cont-oligo or T-oligo+SB samples. (Results are representative from 5 amnion cultures/ group. Telomere mimetic overhang sequence (T-oligo, [TTAGGG]₂); control oligonucleotides (Cont-oligo, [AATCCC]₂); untreated cells (Control CTR).</p

wsCSE increases ASK1 protein levels.

<p>ASK1 (155 kDa) and β-actin (42 kDa) protein bands were identified in amnion cells. Bar graphs demonstrate densitometric quantitation of Western blot band densities (n = 4) normalized to β-actin levels. *indicates significant differences (p<0.05).</p

wsCSE induces increased ROS levels in primary amnion cells.

<p>Preincubation with the antioxidant N-acetyl cysteine (NAC) prevented ROS accumulation (n = 8). Controls – Untreated amnion cells in culture. Data were significant (p<0.05) for all time points for wsCSE treated cells compared to both control and wsCSE+NAC treated amnion cells.</p

wsCSE induces DNA base and strand damage.

<p>DNA damage was determined by comet assays (n = 4) as described in the text. DNA damage was prevented by treatment with NAC prior to CSE exposure. Comet tail lengths were determined microscopically. Untreated amnion cells (control) had minimal comet or FLARE formation. *indicates significant differences (p<0.05).</p