Research Paper Volume 16, Issue 3 pp 2702—2714

Figure 3. Expression levels of miR-146a, senescent and senescence-associated secretory phenotype (SASP) markers, and senescence-associated β-galactosidase (SA-β-gal) activity in LVmiR-146a-transduiced rat primary tenocytes. (A) QRT-PCR for miR-146a expression levels in LVmiR-scramble-(the control vector) and LVmiR-146a-infected rat tendinopathic tenocytes 72h post-infection. (B) LVmiR-146a- and LVmiR-scramble-transduced tenocytes were stimulated with IL-1β (10 ng/mL) or left unstimulated for 96 h. QRT-PCR for determining IL-6 and COX-2 expression levels. LVmiR-146a- and LVmiR-scramble-transduced tenocytes were stimulated with IL-1β (10 ng/mL) or left unstimulated for 24 h (n = 3). Immunoblotting for TRAF6, IRAK-4, phospho-NF-kB, NF-kB, p53, p21, p16, matrix metalloproteinase-1 (MMP)-1 and -3 expression levels in LVmiR-146a- and LVmiR-scramble-transduced tenocytes. (C) Representative figures of immunofluorescence staining for high mobility group box-1 (HMGB1) expression. LVmiR-146a- and LVmiR-scramble-transduced tenocytes were stimulated with IL-1β (10 ng/mL) or left unstimulated for 96 h. DAPI indicates nuclear staining. Scale bars shown at ×40 magnification correspond to 20 μm. (D) The tenocytes were subjected to subjected to SA-β-gal activity assay. SA-β-gal was identified and counted in five high-power fields (200×) to determine the average percentages of SA-β-gal-positive cells corresponding to total cells (n = 4). Results are representative of at least two independent experiments. Values are represented as the mean ± SEM. *p<0.05, ***p<0.001.