Df6285

Total cellular protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (Beyotime) (see Table 2). Cytoplasmic and nuclear proteins were separately extracted according to the manufacturer's instructions using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). Subsequently, the proteins (12 μg) were separated on 10–12% (w/v) SDS-polyacrylamide gel (Beyotime) and transferred onto a PVDF membrane. The membrane was blocked with 5% (w/v) fetal bovine serum (BSA) for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies specific for ETV2 (ab181847, 1:1000, Abcam), OSX (ab209484, 1:1000, Abcam), RUNX2 (ab76956, 1:1000, Abcam), OPN (NB110-89062, 1:1000, Novus), Collagen 1 (ab255809, 1:1000, Abcam), HIF-1α (BF8002, 1:1000, Affinity), PHD1 (EGLN2, DF7918, 1:1000, Affinity), PHD2 (DF6285, 1:1000, Affinity), PHD3 (DF12694, 1:1000, Affinity), VEGFA (66828-1-Ig, 1:1000, Proteintech), ERK1/2 (YT1625, 1:1000, ImmunoWay), pERK1/2 (YP1055, 1:1000, ImmunoWay), VE-Cadherin (AF6265, 1:1000, Affinity), AIF (sc-13116, 1:1000, Santa), Mitofilin (sc-390,707, 1:1000, Santa), and β-actin (ZSGB-Bio). The samples were then incubated with a secondary antibody (1:10,000, ZSGB-Bio) for 1 h and visualized using an ECL kit (Millipore).

The tissue samples were processed as follows: they were initially fixed in 4% paraformaldehyde for 24 h, then embedded in paraffin, and subsequently sectioned into 5-μm thick slices. For hard tissues, extensive decalcification was conducted using 14% EDTA solution (pH 7.4) for four weeks before embedding.
Hematoxylin-Eosin (H&E) and Masson staining were conducted on all serial sections. For immunofluorescence, sections or cells were incubated with primary antibodies, including ETV2 (ab181847, 1:500, Abcam), CD90 (105,201, 1:100, Biolegend), pERK1/2 (YP1055, 1:100, ImmunoWay), HIF-1α (BF8002, AF1009, 1:100, Affinity), PHD2 (DF6285, 1:100, Affinity), CD31 (AF6191, 1:100, Affinity), OPN (NB110-89062, 1:100, Novus), and Collagen I (AF7001, 1:100, Abcam) overnight at 4 °C. Subsequently, secondary antibodies, including FITC-Conjugated Goat anti-mouse IgG (HA1003, 1:200, HUABIO), Alexa Fluor 594 Anti-rabbit IgG (8889, 1:500, Cell Signaling Technology), Alexa Fluor 488 Anti-rabbit IgG (4412, 1:500, Cell Signaling Technology), and FITC-conjugated goat anti-rabbit IgG (SA00003-11, 1:100, Proteintech), were applied for 1 h. Nuclei were labeled with DAPI (Beyotime) for 5 min and images were captured using a fluorescence microscope (Leica).