The expression of the surface epitopes of hADSCs as analyzed by flow cytometry.

<p>Each antibody was tested individually and the isotopes controls were used as the negative control in this experiment; the ADSCs were positive for CD44 (80.2%) and CD73 (93.1%), and negative for CD31 (0.02%) and CD34 (0.03%). For CD44 and CD73, the isotype control was mouse IgG2b and for CD31 and CD34, the isotype control was mouse IgG1.</p

Sequences of primers used in <i>hTERT</i> gene expression, absolute telomere assay and methylation specific PCR.

<p>Sequences of primers used in <i>hTERT</i> gene expression, absolute telomere assay and methylation specific PCR.</p

The schematic figure for showing the 45 CpG sites of the <i>hTERT</i> promoter.

<p>The schematic figure for showing the 45 CpG sites of the <i>hTERT</i> promoter.</p

In vitro multilineage differentiation of hADSCs.

<p>(A) Osteogenic differentiation and alizarin red staining of mineralized cell aggregates; (E) Detection of the two bone specific genes (ALP and OCN) by RT-PCR method (B) Generation of lipid vacuoles after adipogenesis; (F) Expression of PPAR-α and PPAR-γ as fat-specific genes; (C) After chondrogenesis of hADSCs, proteoglycan aggregates stain with toluidine blue; (G) RT-PCR analysis of aggrecan and collagen II as chondrocyte-specific genes; (D) The neuronal cells differentiated from hADSCs show extensive somata-associated accumulations of Nissl bodies stained dark black-violet; (H) Expression of nestin and NGF as neural-specific genes (bar = 40X).</p

Absolute telomere length measurement of hADSCs in the presence of different concentration of ZnSO₄ for 48 hours of incubation.

<p>Cells were seeded at a density of 5×10⁴ cells/wells for about 48 hours in the presence of 1.5×10⁻⁸ and 2.99×10⁻¹⁰ M ZnSO₄. Following, Genomic DNA was isolated, telomere and single copy gene standard curve was created. Real-time PCR technique was used to measure the absolute telomere length. The data were analyzed as kb/reaction and the genome copies/reaction for the telomere and the SCG. As described in results section, 1.5×10⁻⁸ M ZnSO₄ were significantly increased the telomere length of hADSCs (**P<0.01 compared with control group), this procedure was repeated for three times. Data represent as the means ± SE from three independent biological experiments. One-way ANOVA followed by Dunnett’s post hoc test was used to determine the significant difference among groups.</p

Methylation of the <i>hTERT</i> promoter region from 500 bases upstream into the first exon before and after treatment with various concentrations of ZnSO₄.

<p>The CpG sites are numbered from 1–45, CpG sites are indicated as follows: methylation (M); unmethylation (U); not determined (-).</p

Relative <i>hTERT</i> gene expression levels of hADSCs in the presence of ZnSO₄ for 48 hours of incubation.

<p>Cells were seeded at a density of 1.5×10³ cells/wells for about 48 hours in the presence of 1.5×10⁻⁸ and 2.99×10⁻¹⁰ M ZnSO₄. Following, total RNA was isolated and was subjected to Real-time PCR Relative mRNA level of <i>hTERT</i> in the presence of 1.5×10⁻⁸ and 2.99×10⁻¹⁰ M ZnSO₄. As described in results section, 1.5×10⁻⁸ M ZnSO₄ was significantly increased the <i>hTERT</i> gene expression of hADSCs (*P<0.05 compared with control group). This procedure was repeated for three times. Data represent as the means ± SE from three independent biological experiments. One-way ANOVA followed by Dunnett’s post hoc test was used to determine the significant difference among groups.</p

Zinc sulfate contributes to promote telomere length extension via increasing telomerase gene expression, telomerase activity and change in the <i>TERT</i> gene promoter CpG island methylation status of human adipose-derived mesenchymal stem cells - Fig 13

<p><b>ZnSO</b>_<b>4</b><b>effect on (A) ALP, (B) OCN, (C) PPAR-α and (D) PPAR-γ mRNA expression in hADSCs.</b> Cells were cultured in 6-wells plates at a concentration of 30 × 10⁴ cells /wells. RNA was extracted from cultured hADSCs in the presence and absence of 1.5×10⁻⁸ and 2.99×10⁻¹⁰ M ZnSO₄ as described in materials and methods section and was subjected to Real-time PCR assay (*P<0.05 compared with control group). This procedure was repeated for three times. Data represent as the means ± SE from three independent biological experiments. One-way ANOVA followed by Dunnett’s post hoc test was used to determine the significant difference among groups.</p

Relative telomerase activity measurement of hADSCs in the presence of different concentration of ZnSO₄ for 48 hours of incubation as detected by the PCR-ELISA TRAP assay.

<p>Cells were seeded at a density of 5×10⁴ cells/wells for about 48 hours in the presence of 1.5×10⁻⁸ and 2.99×10⁻¹⁰ M ZnSO₄. Following, cells were lysed and protein were extracted from each sample. Relative telomerase activity was assessed in protein extracts by Telomerase PCR-ELISA kit. Heat inactivated cell extract served as the negative control. As described in results section, 1.5×10⁻⁸ M ZnSO₄ was significantly increased the relative telomerase activity of hADSCs (*P<0.05 compared with control group). This procedure was repeated for three times. Data represent as the means ± SE from three independent biological experiments. One-way ANOVA followed by Dunnett’s post hoc test was used to determine the significant difference among groups.</p

Flow chart of an overview of the experimental procedures that have been done in this paper.

<p>Flow chart of an overview of the experimental procedures that have been done in this paper.</p