Mmp 1

The proteins of each group were collected, and the precipitate was separated by lysis, shock, and centrifugation. The precipitate was separated by electrophoresis in 10% SDS-PAGE gel then transferred to PVDF membrane. Following 5% non-fat milk or BSA blocking, the membranes were incubated with related primary antibodies at 4°C overnight. Then, the membranes were incubated with secondary antibodies for 1 h and imaged by ECL chemiluminescence kit (Bio-Rad). The antibodies utilized as follows: MMP1 (1 : 1000; ab137332; Abcam), Vimentin (1 : 2000, #5741, CST), Snail (1 : 1000, #3879, CST), E-cadherin (1 : 2000, #3195, CST), N-cadherin (1 : 1000, #13116, CST), and GAPDH (1 : 2000 dilution, #2118, CST).

Cartilage tissue and chondrocytes were homogenized in a lysate containing protease inhibitors and protein extraction was performed as previously described [16 (link)].
Western blotting was performed according to the manufacturer’s instructions. The primary antibodies used were as follows: NLRP3, ASC, thioredoxin-interacting protein (TXNIP), and caspase-1 (1:1000, Abcam, Cambridge, MA, USA); MMP-1, MMP-13, collagen II, and Unc-51-like kinase 1 (ULK1) (1:2000, Abcam, Cambridge, MA, USA);p-NF-κB and inhibitor kappa B-alpha (IκB-ɑ) (1:500, Cell Signaling Technology, Danvers, MA, USA); and Beclin-1, light chain 3 (LC3), GAPDH, and β-actin (1:500–1:2000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Antigen-antibody complexes were visualized using an ECL Western Detection Kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein levels were normalized to the levels of β-actin or GAPDH. ImageJ software was used to quantify the density of each band.

Mouse skin tissues were fixed in 4% paraformaldehyde for 24 h, and embedded in paraffin. Serial sections (4 μm) were mounted onto silane-coated slides and stained with Hematoxylin and eosin (H&E) for general histopathology or Masson’s trichrome to detect collagen, respectively. For MMP-1 immunostaining, the Envision^TM staining kit (DAKO, was used according to the manufacturer’s instructions. Briefly, slides were deparaffinized and rehydrated. Peroxidases and avidines (DAKO, Carpinteria, CA) were blocked at 10 and 5 min respectively. Samples were washed in PBS, incubated with the primary antibody (MMP-1, Abcam, Cambridge, MA, USA), and with the HRP rabbit Envision^TM System (DAKO) for 1 h at room temperature. Antigen–antibody complexes were revealed by DAB (DAKO) and counterstained with Mayer’s hematoxylin. All stained skin specimens were observed and photographed using an optical microscope (Olympus, Tokyo, Japan). To quantify epidermal hyperproliferation following UV exposure, the vertical thickness of the epidermis was measured at five randomly selected locations per slide with 200× magnification (H&E staining). The data were evaluated through the image analysis program Image J 1.52 (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).

Wells of a 24-well culture plate were coated with 20 μg mL^-1 fibronectin (FN) (Sigma) for 1 h at 37°C and then blocked with 1% sterile BSA. Wells were then treated with GzmB (100 nm) in serum/supplement-free Medium 106 (SFM; Cascade Biologics) for 2 h at 37°C. GzmB was inhibited by Compound 20 (50 μm) (Centre for Drug Research and Development, Vancouver, BC, USA) (Willoughby et al., 2002 (link)) for 30 min before primary human fibroblasts (HDFa, Life Technologies) were seeded (1.5 × 10⁵ cells/well in SFM) and incubated for 20 h at 37°C, 5% CO₂. Control wells were prepared by incubating equivalent amounts of GzmB in SFM in a separate plate (previously blocked with 1% BSA) and then inhibiting with Compound 20. The GzmB/inhibitor media solution was transferred to wells that had been previously coated with FN and blocked as described above, before seeding of cells. After 20 h, supernatants were collected, protease inhibitor cocktail (Sigma) was added, and cell debris was removed by centrifugation. Supernatants were concentrated using centrifuge filters (10 kDa) so that the whole supernatant could be separated as a single sample on a 10% SDS-PAGE gel, transferred to nitrocellulose membranes, and probed for MMP-1 (1/1000, Abcam) or MMP-3 (1/1000, Abcam). Cell lysates from the same wells were collected and probed for GAPDH.