Ab108349

Six micrometers cryosections were obtained from skin biopsies and prepared on poly-L-lysine coated glass slides (Cytospin, Thermo Scientific). Staining for telomere-associated ɣH2AX foci (TAF) was then performed. Sections were stained for ɣH2AX (Ser139, Cell Signaling #9718, 1:250), p16^INK4a (Abcam, ab108349, 1:100 or Sigma SAB5300499, 1:100) and HLA-E (clone 3D12, eBioscience, 1:100), followed by incubation with secondary antibody conjugated to various fluorochromes and washed in formamide/SSC prior to mounting with Vectorshield/DAPI (Vector Laboratories). Slides were air dried prior to hybridisation for 2 h with 40 pM PNA probe targeting the TelC telomeric repeat (Panagene, TelC Cy3, #14 1224PL-01). Imaging of TAF foci was then performed using a Leica SPE2 confocal microscope (Leica Microsystem). Imaging consisted of obtaining Z-stacks with a step-size of 0.5 µm. Analysis was performed using Fiji image analysis software (Fiji.sc).

Human articular cartilage specimens were ground in liquid nitrogen. The cartilaginous surface of dissected mouse knee joints was sliced by a fine knife under a stereomicroscope, collected and ground in liquid nitrogen. The sample for each genotype was obtained from 3–10 mice. Primary antibodies against PTEN (Cell Signaling, 9 188, 1:1 000), phospho-Akt (Ser473) (Cell Signaling, 4 060, 1:1 000), Akt (pan) (Cell Signaling, 4 685, 1:1 000), phospho-mTOR (Ser2448) (Cell Signaling, 5 536, 1:1 000), phospho-FoxO1 (Thr24)/FoxO3a (Thr32) (Cell Signaling, 9 464, 1:1 000), FoxO1 (Cell Signaling, 2 880, 1:1 000), p53 (Cell Signaling, 2 527, 1:500), p16 (Abcam, ab108349, 1:500) and GAPDH (Sigma-Aldrich, G8795, 1:5 000) were employed for Western blot analyses. OxyBlot was operated according to the Millipore OxyBlot^TM protein oxidation detection kit protocol (Millipore, S7150). Western blot results were quantified by densitometry using the ImageJ program. The value for each protein was normalized by its corresponding GAPDH level. The normalized protein level in each control or sham group was then set as 1.0 (100%), and the littermates were presented as the ratio to the corresponding control or sham group.