Batf2

Antibodies used in this study were as follows: BATF2 (ab157466, Abcam, London, UK); BATF2 (Thermo Fisher, PA5-43094); BATF2 (241887, Santa Cruz Biotechnology, CA, USA); CD63 (Abcam, ab217345); CD9 (Abcam, ab92726); TSG101 (Abcam, ab125011); SDF-1α (Abcam, ab9797); SDF-1α (Abcam, ab181018); HIF-1α (H6536, Sigma); CXCR4 (Abcam, ab135170); VEGF (ab46154, Abcam); CD33 (Abcam, ab203253); CD14 (Abcam, ab203294); Gr-1 (108448, Biolegend); CD11b (Abcam, ab13357); CXCR7 (Biolegend, #391405); SV40 Large T Antigen (CST, #15729, CA, USA); MMP-9 (CST, #13667); 10 nm gold-conjugated goat anti-rabbit secondary antibody (bs-0437P-Gold, Bioss, China), GAPDH (sc365062, Santa Cruz Biotechnology), and β-Actin (sc-47778, Santa Cruz Biotechnology).

Immunohistochemistry (IHC) staining was carried out using tumour samples from nude mice or patients as previously described.²², ²⁵ The samples were incubated with primary antibodies against BATF2 (Abcam), CRM1 or Ki67 (Santa Cruz), followed by the corresponding secondary antibodies (Zhongshan Biotechnology, Beijing, China). The staining images were observed using Olympus IX81 photomicroscope. Scoring of IHC staining was performed in accordance with the immunoreactive score, calculated by using percentage of positive cells multiplied by staining intensity.²² Percentage of positive cells—negative: 0; 10%: 1; 11%−50%: 2; 51%−80%: 3; and >80%: 4. Staining intensity—negative: 0; weak: 1; moderate: 2; and strong: 3. The IHC signals were evaluated independently by three pathologists who were blinded to the patients' information.

Western blot was performed as we described (24 (link)). Briefly, proteins were extracted from sarcoma cells and tissues by using RIPA lysis buffer (Beyotime). Proteins were separated and transferred onto PVDF membranes, which were then incubated with the antibodies against BATF2 (Abcam, Cambridge, MA, USA) or β-actin (Santa Cruz, Dallas, TX, USA), followed by the incubation of secondary antibody. Gray-scale of the bands were quantified by using ImageJ software.