Mir 140 mimic

In vitro chondrocyte senescence was induced by 24-h IL-1β (MCE, Pudong, Shanghai, China) stimulation of normal chondrocytes.²² (link) A time course of SA-βGal activity and p16^INK4a staining, including time points of 0 h, 24 h, 48 h, 72 h, 5 days, and 7 days after IL-1β treatment at different concentrations (1, 2, 5, and 10 ng/mL), was investigated, and the IL-1β stimulation paradigm was optimized. A 24-h, 5 ng/mL IL-1β treatment was selected for in vitro chondrocyte senescence establishment, and a further 48-h incubation in fresh media was allowed before the final analysis (Figures S2A and S2B). In the experiments using miR-140 mimic (50 nM; RiboBio, Guangzhou, Guangdong, China) or miR-140 negative control (50 nM, scrambled 22 nt with no homology to mammal genome, miR-Scr; RiboBio), transfection was performed using a FECT CP Transfection Kit (RiboBio), following the manufacturer’s protocols, 24 h before other experimental procedures, and the efficiency of transfection was monitored by quantitative RT-PCR, 24 h after transfection. To distinguish senescent and dysfunctional cells, the IL-1β-treated chondrocytes were also stimulated with FGF2 (1 ng/mL and 10 ng/mL; MCE) and incubated for 48 h for cell cycle analysis.

Cells were transfected with inhibitor of either miR-140 (5′-CAG UGG UUU UAC CCU AUG GUA G-3′), or NC inhibitor (5′-UCA CAA CCU CCU AGA AAG AGU AGA-3′), or miR-140 mimic (5′-CAG UGG UUU UAC CCU AUG GUA G-3′) or NC mimic (5′- UUG UAC UAC ACA AAA GUA CUG-3′) (RiboBio, Guangzhou, China) at 100 nM concentration using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol.