Anti lox 1

OA chondrocytes after treatment with Resveratrol or normal chondrocytes under serum starvation conditions (~200,000 cells) were grown on coverslips in 6-well plates, and were fixed in ice-cold absolute methanol at −20 °C for 10 min, or in 4% paraformaldehyde (PFA) at RT, for 20 min. Fixed samples were then incubated with anti-LC3 (1:200, Cell Signaling Technology, LC3A/B Rabbit #4108), anti-p62 (1:200, Cell Signaling Technology, SQSTM1/p62 Mouse #88588) and anti-LOX-1 (1:100, Santa Cruz Biotechnology, Inc.) and the appropriate fluorescent dye-conjugated secondary antibodies (1:500, Alexa Fluor 594, Molecular Probes) (1:400 Alexa Fluor 488, Molecular Probes). Coverslips were then embedded in 10 μL of Vectashield mounting medium (Vector Laboratories, CA, USA) containing 4,6-diamidino-2-phenylindole (DAPI) to visualize nuclei and observed on a ZEISS Axio Imager.Z2 fluorescent microscope. Images were analyzed using ImageJ software. For calculations, at least 5 randomly selected fields were analyzed for each condition by two independent observers blinded to the origin of the sample (Normal or OA chondrocytes, treated or not treated (Control)). Each observer counted at least 200 cells for each time point and the means of their counts were used for the statistical analysis.

Slides containing serial cross‐sections of thoracic descending aortic tissue or HAECs were immunostained with antiphosphorylated histone H2AX (anti‐γH2AX) antibody (Cell Signaling Technology) and then with Alexa‐488 conjugated secondary antibody. Hoechst 33342 or DAPI (4′,6‐diamidino‐2‐phenylindole) was used to stain nucleic acids and to assess the formation of senescence‐associated heterochromatin foci. Anti–LOX‐1 (Santa Cruz Biotechnology, Dallas, TX), anti‐TP53 (Cell Signaling Technology), anti‐p16^INK4a (Abcam Inc., Cambridge, MA), and anti‐3‐nitrotyrosine (EMD Millipore, Darmstadt, Germany) antibodies were used to determine in situ the abundance of LOX‐1, TP53, p16^INK4a, and 3‐nitrotyrosine in aortic tissue samples. Anti‐hTERT monoclonal antibody (EMD Millipore, Billerica, MA, USA) was used to detect hTERT expression in HAECs. (Klokov, MacPhail, Banath, Byrne, & Olive, 2006). The semi‐quantification of immunofluorescence staining is described in the Supplemental Methods.

Total cell lysates were extracted from keratinocytes. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane. The membranes were probed using anti-Ki67 (Santa Cruz, CA, USA), anti-LOX-1 (Santa Cruz, Dallas, TX, USA), anti-scavenger receptor A (SRA; Santa Cruz, CA, USA), anti-SR-B1 (Santa Cruz, Dallas, TX, USA), and anti-IL23 (Novusbio, Littleton, CO, USA) antibodies. Mouse anti-β-actin (Labvision/NeoMarkers, Fremont, CA, USA) antibody was used as a loading control. The proteins were visualized using an enhanced chemiluminescence (ECL) detection kit (Amersham Biosciences, Waltham, MA, USA) and quantified using a densitometry.