Table 1.
The equine primer sequences used for real-time PCR.
Fig 1.
A. Viability of tenocytes following dexamethasone treatment by the MTT assay. The results are expressed as the mean ± SD of three independent experiments. One-Way ANOVA with Bonferroni’s Multiple Comparison Test was applied. B. Cell proliferation assay following dexamethasone treatment. The percentage of cell cycling after 48 h dexamethasone and etoposide treatment was measured by EdU and Ki67 labelling. The data represent the average and standard deviation of three independent experiments (n = 3 horses) using one-way ANOVA Bonferroni’s multiple comparison test. (ET: etoposide, DEX: dexamethasone) (*p<0.05, **p<0.01, ***p<0.001). C. Cell proliferation assay in serum free medium containing dexamethasone for 48h. The results are expressed as the Mean ± SD of three experiments using One-Way ANOVA with Turkey’s Multiple Comparison Test. EdU and Ki67 labelling were performed in duplicate and at least 500 cells were inspected to determine the percentage of ki67-positive cells(DEX: dexamethasone) (*p<0.05, **p< 0.01).
Fig 2.
Expression of p53, p21 and p16 in dexamethasone-treated tendon derived cells.
Total RNA from TDCs was analysed by qPCR at (2A) 24h and (2B) 72h following dexamethasone (DEX) treatment. P53 was not detected at 72 h (*p<0.05, **p<0.01, ***p<0.001).
Fig 3.
Induction of a SASP in tenocytes by dexamethasone.
Cytokines were assayed in culture medium by ELISA at 5 days after dexamethasone treatment. There were no statically significant differences in IL-8 and MCP-1 levels between the 1 and 10μM concentrations of dexamethasone (DEX).
Fig 4.
The gene expression level of MMP-1, MMP-2 and MMP-13 in dexamethasone-treated TDCs 5 days after removing dexamethasone (DEX) was analysed by qPCR. Data were normalized to GAPDH and expressed as fold change over control levels. One-way ANOVA,* p<0.05,** p<0.01.
Fig 5.
A. EdU and Ki67 labelling for proliferating cells. Statistically significant differences between 1 or 10μM dexamethasone (DEX) and Resveratrol (RSV)+ DEX are indicated by ** p<0.01. B. p53 and p21 genes expression. The expression level of p53 and p21 in dexamethasone and resveratrol-treated(DEX+RSV) TDCs 24h after removing DEX was analysed by qPCR. *p<0.05, **p<0.01.
Fig 6.
A. Quantification of SA-β-gal staining following a 48-hour exposure to 1 and 10μM dexamethasone and 2μM resveratrol (DEX+RSV). Cells were assessed for the percentage of SA-β-gal positive staining seven days post-treatment. SA-β-gal staining was performed in duplicate and at least 100 cells were inspected to determine the percentage of SA-β-gal positive cells. Statistical analysis performed using One-way ANOVA (*p<0.05,**p< 0.01). B. Resveratrol inhibited SASP development in dexamethasone-treated cells. The levels of IL-8 and MCP-1 were measured by cytokine array 5 days after treatment with 1 and 10μM dexamethasone and 2μM resveratrol (DEX+RSV). The values are shown as the mean ± SD. **p<0.01,***p < 0.001. C. The effect of resveratrol on MMPs gene expression in dexamethasone and resveratrol-treated (DEX+RSV) TDCs. The gene expression levels of MMP-1, MMP-2, and MMP-13 were measured by qPCR five days after the removal of treatment. Data were normalized to GAPDH expression and presented as fold change over the control level. Statistical analysis performed using One-way ANOVA (*p<0.05, **p< 0.01, ***p<0.001).
Fig 7.
EdU labelling for proliferating cells.
Statistically significant differences between 1 (A) or 10μM dexamethasone (DEX) (B) and Resveratrol (RSV)/ V34 and V29+ DEX are indicated by * p<0.05 ** p<0.01.
Fig 8.
The effect of different concentrations of SRT-1720 on TDCs proliferation against dexamethasone treatment.
Data are presented as the Mean ± SD of EDU positive cells observed between 1 and 10μM dexamethasone (DEX) and 0.5 and 1μM SRT-1720 (SIRT). Statistical analysis was performed using One-Way ANOVA with Bonferroni’s Multiple Comparison Test (*p<0.05, **p< 0.01) (n = 3).