2.5.4. Quantitative Senescence-Associated Beta-Galactosidase Assay

4-methylumbelliferyl-β-D-galactopyranoside (4-MUG) was used as substrate of βgalactosidase for the quantitative SA-β-gal assay [40]. 4-MUG does not fluoresce until cleaved by the enzyme to generate the fluorophore 4-methylumbelliferone. The assay was carried out on lysates obtained from cells that were grown as reported above. The production of the fluorophore was monitored at an emission/excitation wavelength of 365/460 nm.

#### 2.5.5. RNA Isolation, Reverse Transcription, and Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from cell cultures using TriFast (EuroClone, Milan, Italy), according to the manufacturer's protocol, and mRNA levels quantified by RT-PCR amplification as reported by Calarco el al. [41]. For retro-transcription, total RNA (0.5 μg) was treated as described in EuroClone standard protocol and amplified by qPCR. Specific primers for SRY-Box Transcription Factor 9 (*SOX9*), Collagen Type II Alpha 1 Chain (*COL2A1*), Aggrecan (*ACAN*), Cartilage Oligomeric Matrix Protein *(COMP*), Interleukin-6 (*IL-6*), Interleukin-8 (*IL-8*), tumor necrosis factor (*TNF*)-*α*, Matrix Metallopeptidase 3 and 13 (*MMP-3* and *13*), and β-Actin (*ACTB*) were used and listed in Table 1. qRT-PCR was run on a 7900 HT fast real-time PCR System (Applied Biosystem, Milan, Italy). The reactions were performed according to the manufacturer's instructions using SYBR Green PCR Master mix (Euroclone, Italy). Data were normalized using the housekeeping gene (ACTB). All reactions were run in triplicate and the results expressed as mean <sup>±</sup> SD. The 2−ΔΔCt method was used to determine the relative quantification.

**Table 1.** Primers used for qRT-PCR.

