Sc 30833

The western blotting experiments were performed as previously described [26 (link)]. The following antibodies were used: mouse monoclonal anti-GAPDH (1:4 000 dilution, G8795, Sigma-Aldrich), anti-HYAL1 (1:1 000 dilution, 1D10, produced by hybridoma cells generously provided by B. Triggs-Raine, University of Manitoba, Winnipeg, Canada) and anti-cathepsin K (1:1 000 dilution, MAB3324, Millipore), as well as goat polyclonal anti-TRAP (1:1 000 dilution, SC-30833, Santa Cruz Biotechnology). When conditioned media were prepared, the cells were cultured for 5 h in serum-free medium prior to lysis of the cells in PBS—Triton X-100 1% supplemented with protease inhibitors (cOmplete, mini protease inhibitors cocktail, Roche). When indicated, cells were incubated for 24 h with either 15 mg/mL of mannan (Sigma-Aldrich) or 5 mM of Man-6-P (Sigma-Aldrich). In Fig 5C, cell extracts and conditioned media were treated with PNGase F (New England Biolabs) according to the manufacturer's instructions. Of note, the specificity of the anti-HYAL1 antibody was validated by an absence of signal in osteoclasts differentiated from BMM of Hyal1 -/- mice (S1A Fig). HYAL1 signals were quantified using the ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2015).

For cell immunofluorescence staining, cells were seeded on 3.5 cm confocal dish at the density of 3 × 10⁴. Cells were treated with or without the autophagy-flux inhibitor chloroquine (CQ) (50 μM) 3–5 h before staining. In sequence, cells were fixed with 4% paraformaldehyde at 4 °C for 10–15 min, washed with PBS, incubated with 0.5% Triton-100 at room temperature for 10 min, and blocked with PBS containing 1% BSA at room temperature for 40 min. Next, the samples were incubated with primary antibodies to LC3 (Cell Signaling Technology, 1274, 1:100), aggrecan (GeneTex, GTX54920, 1:100), collagen II (Abcam, ab34712, 1:100), OCN (Santa Cruz Biotechnology, sc-390877, 1:100), PPAR-γ (Abcam, 2435, 1:50), and TRAP (Santa Cruz Biotechnology, sc-30833, 1:100) overnight at 4 °C and subsequently incubated with fluorescent secondary antibodies, respectively. The positive cells were examined under a laser scanning confocal microscope (Olympus FluoViem FV 1000, Tokyo, Japan). Quantitative histomorphometric analysis was conducted with Image Pro Plus software.