Sc 1661

VSMCs, macrophages and proliferating cells were identified by immunohistochemistry for α-smooth muscle cell actin (Sigma, A2547, 3.1 µg/ml)and desmin (R&D Systems, AF3844, 5 µg/ml), Griffonia Simplicifolia Lectin-1 (GSL, Vector, B-1205, 2.5 µg/ml) and CD68 (LSBio, LS-C343891, 1 µg/ml) and proliferating cell nuclear antigen (PCNA, abcam 18197, 1 µg/ml) and Ki67 (DAKO, M7249, 3.8 ug/ml), respectively, as described previously^{14 (link),24 (link)}. Cleaved caspase-3 (R&D Systems, AF835, 10 µg/ml) as described previously^{14 (link),24 (link)}) and cleaved PARP (abcam, ab32064, 4.8 µg/ml) were used to identify apoptotic cells. Briefly, antigen retrieval was performed by boiling in citrate buffer, blocking step was with 5% goat serum, the primary antibody for cleaved PARP was added and incubated overnight at 4 °C. Biotinylated anti-rabbit secondary antibody was utilised, followed by Extravidin-HRP and DAB (3,3′-diaminobenzidine). Nuclei were counterstained with haematoxylin. Immunohistochemistry for a marker of cellular senescence, p16 (Santa Cruz, sc-1661, 2 µg/ml) was performed as described previously^{27 (link)}. Non-immune immunoglobulin controls were performed for all protocols by substituting the primary antibody with non-immune immunoglobulin of the same species and at the same concentration (Supplementary Figure 3).

Mouse skin was homogenized in ice cold lysis buffer (50 mm Tris–HCl, pH 7.5, 150 mm NaCl, 0.5% NP40, 20% glycerol) with freshly added proteases and phosphatases inhibitor cocktails (Roche, Vienna, Austria). The homogenate was centrifuged at 15 000 g for 30 min at 4°C. The supernatant was collected and the aliquots were stored at −70°C. The protein content was measured by Bradford assay. Equal amounts of protein were subjected to electrophoresis on 4–12% NuPage gels (Invitrogen, Carlsbad, CA, USA) in 1× MOPS running buffer and then transferred to PVDF membrane. The PVDF membranes were subsequently blocked with 3% non‐fat milk in 1% Tween 20–PBS buffer for 1 h at room temperature. PVDF membranes were incubated with the following primary antibodies: Pso4/Prp19 (SNEV) (1:1000, A300‐102A, Bethyl Lab); γ‐H2AX (1:1000, ab26350; Abcam, Cambridge, UK); p16^INK4a (1:200, SC‐1661; Santa Cruz); MMP‐13 (1:500, ab75606; Abcam); beta‐actin (1:10 000, A5441; Sigma‐Aldrich); and gapdh (1:1000, sc‐25778; Santa Cruz) overnight at 4°C. Thereafter, incubation with secondary antibodies Alexa 680 – (1:10 000; Life Technologies, Carlsbad, CA, USA) or Infrared 800‐conjugated (1:10 000; Lincoln, Nebraska, USA) was performed for 1 h at room temperature. Detection was performed using an Odyssey Infrared scanner, and the optical density of the bands was measured using Image Studio software vs. 2.1 (Licor).

Total protein was extracted from mouse skin tissues using ice-cold RIPA lysis buffer (Beyotime) containing a protease inhibitor mixture (Roche). The total protein content was quantified using the BCA method. Protein samples were subjected to Mini-PROTEAN^® TGX Stain-Free™ Precast 10% or 12% gels (Bio-Rad) and transferred to 0.2 µm PVDF membranes (Millipore) using the Trans-Blot Turbo Transfer Pack and Trans-Blot Turbo Transfer System (Bio-Rad). PVDF membranes were incubated with primary antibody overnight at 4°C, followed by HRP-coupled secondary antibody for 1 h at room temperature. Bio-Rad ChemDoc MP was used for Western blot data collection and analysis. The primary antibodies used in this study were Tgf-β (1:1000; ab215715, Abcam), IL-6 (1:1000; ab9324, Abcam), γ-H2AX (1:1000; ab81299, Abcam) and p16 (1:1000; sc1661, Santa Cruz).