Runx2

Antibodies against runt-related transcription factor-2 (RUNX2), osteopontin (OPN), NADPH oxidase1 (NOX1), and NADPH oxidase2 (NOX2) were purchased from Abcam (Cambridge, UK). Cu/Zn superoxide dismutase (Cu/Zn SOD or SOD1), catalase (CAT), NF-κB signaling protein (p65, phosphor-p65, and IκBα), goat anti-mouse IgG, and goat anti-rabbit IgG secondary antibodies were from Cell Signaling Technology Inc. (Boston, MA, USA). Abs against GAPDH was from Proteintech Group Inc. (Rosemont, IL, USA). Melatonin was purchased from Sigma-Aldrich (St. Louis, MO, USA), and recombinant human TNF-α was purchased from R&D Systems (Minneapolis, MN, USA). The osteogenic differentiation medium was purchased from Cyagen Biosciences Inc. (Santa Clara, CA, USA).

FFPE tibia bones were further sequentially sectioned and baked at 56°C in preparation for immunofluorescence staining of osteoblast (RUNX2 at 1:500; Abcam Cat. No. ab81357) and nuclear staining (DAPI). Deparaffined and rehydrated slides were subject to heat-induced antigen retrieval method. Sections were then blocked and incubated in primary antibodies diluted in 10% normal goat serum in TBS overnight at 4°C. Subsequently, slides were stained with secondary Alexa Fluor 568-conjugated antibody at 1:1000 at room temperature for 1 hour under light-proof conditions. Stained slides were stained with DAPI for nuclear contrast and mounted for imaging at 20X using Zeiss upright fluorescent microscope to include the injury site as well as the immediate peripheral tissue. All RUNX2 positive cells (red staining colocalizing with DAPI) within 5μm radius from injury were counted and mathematically converted to osteoblasts / bone marrow volume (#OBL/μm³) for each bone at each time point.

Antibodies against FNDC5 (amino acids 50–150), Runx₂, were purchased from Abcam (Cambridge, MA, USA). Anti-FLAG, Anti-GAPDH, and HRP-conjugated secondary antibodies were obtained from Zen-Bioscience Company (Chengdu, China). Antibodies against phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, ERK, p-ERK, P38, p-P38, JNK, p-JNK, GSK-3β, p-GSK-3β, and β-catenin were purchased from Cell Signaling Technology (Waltham, MA, USA). dexamethasone, ascorbic acid, β-glycerophosphate and Hexadecylpyridinium Chloride Monohydrate were purchased from Sigma (Louis, MO, USA). r-irisin (067-16) and irisin-ELISA Kit (EK-067-29) were purchased from Phoenix (Burlingame, CA. USA).

Cells were cultured for 4 d on the stiff or soft gel-coated slides, and fixed with 4% paraformal- dehyde. After blocking in blocking buffer (containing 3% goat serum, 10% horse serum, 0.3% Triton X-100 in PBS) for 30 min at room temperature, the slides were incubated with primary antibodies against Sox9 (1:100; Abcam), Aggrecan (1:50; Abcam), ColII (1:50; Abcam), Runx2 (1:100), ColI (1;100), MAP2 (1:500; Abcam) , Nestin (1:100; Abcam) or NFL (1:500; Abcam), then with anti-mouse IgG conjugated with FITC (1:4,000; Abcam), anti-rabbit IgG conjugated with TRITC (1:4,000; Abcam) or anti-goat IgG conjugated with TRITC (1:4,000; Abcam). Finally, cell nuclei were visualized with DAPI (Sigma-Aldrich, St. Louis, Missouri, USA) and viewed under a Leica TCS SP5 confocal microscopy system (Leica, Germany).

Details of the Western blot protocol are described elsewhere^{36 (link)}. Briefly, total protein was collected using cell lysis reagent containing a protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Protein samples were boiled for 5 min, loaded onto a 10% SDS-PAGE gel for separation, and transferred onto polyvinylidene fluoride (PVDF) membranes at 300 mA for one hour. After blocking with 5% bovine serum albumin (BSA) for 2 hours, the membrane was incubated overnight at 4 °C with primary antibodies against GATA4 (Abcam, USA), dentin sialophosphoprotein (DSPP, Abcam, USA), runt-related transcription factor 2 (RUNX2, Abcam, USA), osterix (OSX, Abcam, USA), osteopontin (OPN, Abcam, USA), osteocalcin (OCN, Abcam, USA), bone morphogenetic protein 4 (BMP4 Abcam, USA), GNAI3 (Abcam, USA) and GAPDH (Bioworld, China). Subsequent to this, the membranes were incubated with secondary antibodies at room temperature for 1 hour, rinsed with Tris Buffered Saline (with Tween-20) three times, and visualized by enhanced chemiluminescence. Semi-quantitative measurements were carried out using Image J software (National Institutes of Health, USA).

The de-paraffinized knee joint sections from DMM and sham mice were incubated overnight at 4° C with primary antibodies against BMP5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Runx2, Col2a, MMP13, Adamts5 (Abcam, Cambridge, UK), as well as p16 and p21 (Proteintech, Rosemont, IL, USA). The slides were then incubated with the secondary antibody (Jackson ImmunoResearch Laboratories, Baltimore Pike, USA, 1:200)) in blocking solution for 1 h at room temperature, and developed with 3,3′-diamino-benzidine (DAB, ZSGB-Bio, Beijing, China).