Effect of zinc sulphate (ZnSO₄) on the proliferation of rat Adipost tissue-MSCs (rADSCs) (^*P<0.05 vs. control, mean ± SEM; n = 3).

<p>rADSCs were cultured at the density of 2 × 10³ cells per well in the absence of ZnSO₄ and in the presence of 4.32, 0.432, 0.0432 and 0.00432 μg/ml of ZnSO₄, as described in section 2.4. MTT dye solution was added when rADSCs were cultured for 21 days. After 4 h incubation, culture medium was removed, DMSO was added and the optical density of each well was measured at a wavelength of 570 nm.</p

ALP, OCN, Runx2 and BMP2 are regulated by PKA and ERK1/2.

<p>The ALP activity of rat Adipost tissue-MSCs (rADSCs) treated with 50 Hz, 20 mT EMF in the presence and absence of 0.432 μg/ml ZnSO₄ in groups I, II, III and IV as described in section 3.7 alone and plus (A) H-89 at the 10μM concentration and plus (B) PD98059 at the 20μM concentration for 6 days; Data are shown as mean ± SEM; ^*P<0.05, ^**P<0.01. (C) OCN, (D) Runx2 and (E) BMP2 were regulated by EMF and ZnSO₄ requiring PKA and ERK1/2 signaling pathways. The cells were subjected to 21 days of 50 Hz, 20 mT EMF treatments in the presence and absence of 0.432 μg/ml ZnSO₄ in groups I, II, III and IV as described in section 3.7 for 30 min/day, with 10μMH-89 or 20μMPD98059 added to the cells. RNA was extracted from cultured rADSCs in groups I, II, III and IV and was subjected to Real-time PCR assay; mean ± SEM; n = 3; ^*P<0.05, ^**P<0.01.</p

Enhancement of osteogenic differentiation of rat adipose tissue-derived mesenchymal stem cells by zinc sulphate under electromagnetic field via the PKA, ERK1/2 and Wnt/β-catenin signaling pathways - Fig 5

<p><b>(A) ALP activity and (B) calcium levels were affected by EMF and ZnSO</b>_<b>4</b><b>.</b> Cells were cultured in 24-well plates containing an osteogenic induction medium as described in section 2.6 and 2.7 at a density of 10 × 10⁴ cells/well. For ALP activity measurement, protein was extracted and for calcium assay, ultrasonication was done from the cultured rADSCs on days 4, 8, 16 and 20 post-EMF treatment with 50 Hz, 20 mT and post-ZnSO₄ treatment in the concentration of 0.432 μg/ml for 30 min/day for 21 days. Likewise, protein extraction was performed on the controls; mean ± SEM; n = 3; ^*P<0.05, ^**P<0.01, ^***P<0.001.</p

In vitro multilineage differentiation of rat Adipost tissue-MSCs (rADSCs).

<p>(A) Osteogenic differentiation and cell aggregates (were stained with alizarinred staining). Arrows show some of the mineralized cell aggregates (bar = 200μm). (B) Differentiation into adipose cells. Arrows show lipid vacuoles generated after adipose differentiation (bar = 200μm). (C) Differentiation of rADSCs to chondrocytes, proteoglycan aggregates stain with toluidine blue (bar = 200μm). (D) The neuronal cells differentiated from rADSCs show extensive somata-associated accumulations of Nissl bodies stained dark black-violet (black arrows) (bar = 200μm).</p

Flow cytomertic analysis of rat Adipost tissue-MSCs (rADSCs).

<p>rADSCs are stained with monoclonal antibodies directed against either CD44, CD90, CD105, CD31, CD34 and CD56 and coupled to fluorescein isothiocyanate (FITC); these signals are indicated as red lines, An isotype matched monoclonal antibody served as a control; these signals are indicated as dotted lines. rADSCs were positive for CD44, CD90 and CD105 and negative for CD31, CD34 and CD56.</p

Spindle-shaped morphology of rat Adipost tissue-MSCs 7 day after seeding (bar = 200μm).

<p>Spindle-shaped morphology of rat Adipost tissue-MSCs 7 day after seeding (bar = 200μm).</p

Primer sequences used for the real-time PCR assays.

<p>Primer sequences used for the real-time PCR assays.</p

cAMP and PKA activities affected by EMF and ZnSO₄.

<p>Cells were exposed to 50 Hz, 20 mT EMF in the presence and absence of 0.432 μg/ml ZnSO₄ in groups I, II, III and IV as described in section 2.8 and 2.9. After the termination of EMF exposure at the end of the 21st day, the plates were kept in the incubation chamber (A) for 5, 15, 30, 45, 60, 90 and 120 min before cells were collected for cAMP analysis, and (B) for 30, 60, 90 and 120 min before PKA activity assay; mean ± SEM; n = 3; ^*P<0.05, ^**P<0.01.</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