Sp7 osterix

Proteins were extracted using RIPA buffer (Beyotime) supplemented with 1% phosphatase inhibitor and 1% phenylmethanesulfonyl fluoride. Proteins were loaded and separated on 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes for immunoblotting. The following antibodies were used: GAPDH (1/10000; Abcam, Cambridge, United States ), Runt-related transcription factor 2 (Runx2, 1/1000; Abcam), osterix (Sp7, 1/1000; Abcam), type I collagen (COL1, 1/1000; Abcam), and β-catenin (1/1000; CST, United States ). The membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1/10000; GenScript, United States ) for 1 h. The blot was scanned on the GelView 6000PLus Smart Gel Imaging System (BLT, Guangzhou, China). The quantification of protein levels was calculated as a ratio of the protein of interest to that of the loading control (GAPDH).

HBMSCs and mBMSCs were provided by Cyagen Biosciences (HUXMA-01001, MUBMX-01001, Guangzhou, China), which can differentiate into osteoblasts, chondrocytes, and adipocytes under specific inductive conditions. Adherent hBMSCs were incubated in culture flasks in a special complete growth medium (HUXMA-90011, Cyagen Biosciences, Inc., Guangzhou, China) in a cell incubator at 37 °C with 5% CO₂ and were passaged at nearly 80–90% confluence. Cells from passages two to six were used in subsequent experiments.
Specific antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and FOXA1 were purchased from PROTEINTECH (Chicago, USA). Runt-related transcription factor 2 (RUNX2), extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2 (p-ERK1/2) were obtained from Cell Signalling Technology (Danvers, MA, USA). Specific antibodies against collagen type I alpha 1 (COL1A1) and Osterix (SP7) were purchased from Abcam (Cambridge, UK) and PROTEINTECH (Chicago, USA). A phospho-p44/42 MAPK (P-ERK1/2) inhibitor, PD98059, was purchased from MedChemExpress (New Jersey, USA).

To prepare the paraffin-embedded histology sections, samples were decalcified with 10% EDTA for 3 weeks at room temperature after image collection by micro-CT. Sections were stained with H&E for morphological analysis.
EdU-incorporated proliferative cells were detected using the Click-iT EdU Cell Proliferation Kit for Imaging Alexa Fluor 488 dye (Invitrogen) according to manufacturer instructions. Following the detection of EdU-labeled cells, molecular markers were co-labeled by immunohistochemistry, as previously described^{40 (link)}. The primary antibodies used were as follows: mCherry (1:2,000; M11217; Invitrogen), Gli1 (1:400; 78259; NOVUS, Littleton, CO, USA), Ring1b (1:100; 101273; Abcam, Cambridge, UK), Cbfa1/Runx2 (1:400; 035; MBL Life Sciences, Nagoya, Japan), Sp7/Osterix (1:800; 22552; Abcam), and PCNA (1:200; 2586; Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies were as follows: goat anti-rabbit IgG antibody Alexa Fluor 555 (1:400; Invitrogen) and goat anti-mouse IgG antibody Alexa Fluor 555 (1:400; Invitrogen). For the detection of Gli1, Alexa Fluor 555 Tyramide SuperBoost Kit goat anti-rabbit IgG (Invitrogen) was used to amplify the immunoreactivity according to manufacturer instructions. The samples were enclosed with VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA).