Ma5 17142

Duolink PLA in situ fluorescence (Sigma-Aldrich) was performed according to the manufacturer’s instructions with Duolink in situ PLA probe anti-mouse PLUS (#DUO92001), Duolink in situ PLA probe anti-rabbit MINUS (#DUO92005), Duolink in situ detection reagents Red (#DUO92008) and Duolink in situ wash buffers-fluorescence (#DUO82049). Pulmonary fibroblasts from Bmi-1^−/− mice were treated with TGF-β1 and detected with antibodies against p16 (MA5-17142, Thermo Fisher Scientific, IL, USA) and ERK1/2 (#4695, Cell Signaling Technology), and p16 (MA5-17142, Thermo Fisher Scientific, IL, USA) and pERK1/2(Thr202/Tyr204) (#4370, Cell Signaling Technology). The PLA signal (λex 594 nm, λem 624 nm; Texas Red) was analyzed as previously described^{28 (link)}.

Lower limbs isolated from tibia-loaded mice were fixed in 10% neutral buffered formalin for 24 h and transferred into 70% ethanol. Further processing and sectioning were performed at Histoserv (Germantown, MD). Tissue sections were cut at 6-μm thickness with 50-μm space between consecutive sections. Cartilage tissue sections were stained for Safranin O and fast green or used. Immunohistochemistry for Jnk (CST, 9255S; 1:250) and p16 (Thermofisher, MA5-17,142; 1:250) was carried out.
Human cartilage from joint tissues after total knee replacement of patients (under Stanford-approved IRB protocols) was divided into smaller discs using biopsy punches (2-mm diameter). These cartilage discs were then cultured with or without JNK inhibitor for a week before processing. The tissues were embedded in OCT and sectioned in Leica cryotome at 6-μm thickness and stained as described above.