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. 2023 Jan 2;11:3. doi: 10.1038/s41413-022-00243-8

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Fap degrades denatured or MMP13-cleaved Col II. a Fap degrades denatured Col II in a dose-dependent manner. Denatured Col II (boiled at 95 °C for 10 min) was incubated with different amounts of rFap at 37 °C for 24 h. Samples were separated by SDS‒PAGE and quantified by colloidal blue staining (n = 3 independent experiments). b Fap degrades denatured Col II in a time-dependent manner. Denatured Col II was incubated with rFap at 37 °C for 0–24 h (n = 3 independent experiments). c FAPi inhibits Fap-mediated degradation of denatured Col II. Different doses of FAPi were preincubated with rFap for 30 min before denatured Col II was added and further incubated at 37 °C for 24 h (n = 3 independent experiments). d Immunoprecipitation of Fap. HEK293T cells were transfected with GFP control, Oln-Flag, Fap-HA, or Oln-Flag + Fap-HA. Two days after transfection, Fap was immunoprecipitated from total cell lysates with 10 μL anti-HA affinity gel. Five percent total cell lysates were loaded as an input control (n = 3 independent experiments). e Oln inhibits the Fap-mediated degradation of denatured Col II. Col II was coincubated with immunoprecipitated samples (in 10 μL anti-HA affinity gel) at 37 °C for 24 h (n = 3 independent experiments). f Fap degrades MMP13-cleaved Col II. Native Col II was preincubated with rFap or rMMP13 at 37 °C for 12 h. EDTA was then added to the reaction mixture with or without rFap and incubated at 37 °C for another 12 h (n = 3 independent experiments). g Grayscale quantification of the 55, 40 and 30 kDa digestion bands in (f). h Experimental design. Primary chondrocytes were stimulated with vehicle control (PBS), 200 ng·mL⁻¹ rFap, 200 ng·mL⁻¹ rMMP13, or 200 ng·mL⁻¹ rFap plus 200 ng·mL⁻¹ rMMP13 for 24 h. i Western blot analysis of mouse Col2a1 protein levels in primary chondrocytes stimulated as in (h) (n = 3 independent experiments). The statistical significance was assessed using one-way ANOVAs with Tukey’s multiple comparison tests. Data are presented as the mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001)