Figures & data
Figure 1 The expression of ERβ in OS and adjacent normal tissues of 24 OS patients detected by qRT-PCR and IHC. (A) Relative expression of ERβ at mRNA level (qRT-PCR); (B) positive stained cells (Bar =50 μm, ×200). ***P<0.001.
Abbreviations: ERβ, estrogen receptor β; OS, ostemsarcoma; qRT-PCR, quantitative real-time PCR; IHC, immunohistochemistry.
Figure 2 The expression of ERβ in U2-OS and hFOB1.1 cells detected by qRT-PCR and Western blot. (A and C) Relative expression of ERβ at mRNA level (qRT-PCR); (B and D) relative expression of ERβ at protein level (Western blot). si-ERβ, U2-OS cells transfected with siRNA-ERβ for 48 hrs; NC-ERβ, U2-OS cells transfected with siRNA-negative control-ERβ for 48 hrs; blank, U2-OS cells without transfection. **P<0.01; ***P<0.001.
Abbreviations: ERβ, estrogen receptor β; OS, ostemsarcoma; qRT-PCR, quantitative real-time PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; si, small interfering RNA.
Figure 3 The expression of β-catenin in U2-OS and hFOB1.1 cells detected by Western blot and Immunofluorescence. (A and B) Relative expression of β-catenin at protein level (Western blot); (C) positive stained cells (bar =100 μm, ×100). si-ERβ, U2-OS cells transfected with siRNA-ERβ for 48 hrs; NC-ERβ, U2-OS cells transfected with siRNA-negative control-ERβ for 48 hrs; si-ERβ + FH535, U2-OS cells transfected with siRNA-ERβ and treated with 20 μmol/L FH535 for 48 hrs; blank, U2-OS cells without transfection and treatment. **P<0.01; ***P<0.001.
Abbreviations: ERβ, estrogen receptor β; OS, ostemsarcoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4',6-diamidino-2-phenylindole; NC, negative control; si, small interfering RNA.
Figure 4 The migration of U2-OS cells detected by Scratch assay. (A) scratch healing under microscope (Bar =500 μm, ×4); (B) scratch healing rate; (C) migration cells under microscope (bar =200 μm, ×20); (D) number of migration cells. si-ERβ, U2-OS cells transfected with siRNA-ERβ for 48 hrs; NC-ERβ, U2-OS cells transfected with siRNA-negative control-ERβ for 48 hrs; si-ERβ + FH535, U2-OS cells transfected with siRNA-ERβ and treated with 20 μmol/L FH535 for 48 hrs; Blank, U2-OS cells without transfection and treatment. ***P<0.001.
Abbreviations: ERβ, estrogen receptor β; OS, ostemsarcoma; NC, negative control; si, small interfering RNA; h, hours.
Figure 5 The invasion of U2-OS cells detected by Transwell assay and Western blot. (A) Invasion cells under a microscope (bar =200 μm, ×20); (B) the number of invasion cells; (C) protein brands of Western blot; (D) relative expression of MMP-7 and MMP-9 at protein level (Western blot). si-ERβ, U2-OS cells transfected with siRNA-ERβ for 48 hrs; NC-ERβ, U2-OS cells transfected with siRNA-negative control-ERβ for 48 hrs; si-ERβ + FH535, U2-OS cells transfected with siRNA-ERβ and treated with 20 μmol/L FH535 for 48 hrs; blank, U2-OS cells without transfection and treatment. ***P<0.001.
Abbreviations: ERβ, estrogen receptor β; OS, ostemsarcoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMP, matrix metalloproteinase; NC, negative control; si, small interfering RNA.
Figure 6 The growth and metastasis of OS tumors in mice. (A) Subcutaneous tumors under naked eye; (B) subcutaneous tumor volumes at different time points; (C) protein brands of Western blot; (D) relative expression of β-catenin, MMP-7 and MMP-9 at protein level (Western blot); (E) metastatic tumors in the liver tissues of mice under microscope (HE staining) (bar =50 μm, ×40); (F) the number of metastatic tumors. si-ERβ, U2-OS cells transfected with siRNA-ERβ for 48 hrs; NC-ERβ, U2-OS cells transfected with siRNA-negative control-ERβ for 48 hrs; si-ERβ + FH535, U2-OS cells transfected with siRNA-ERβ and treated with 20 μmol/L FH535 for 48 hrs; blank, U2-OS cells without transfection and treatment. ***P<0.001 vs NC-ERβ and blank; ###P<0.001 vs si-ERβ.
Abbreviations: ERβ, estrogen receptor β; OS, ostemsarcoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMP, matrix metalloproteinase; HE, hematoxylin-eosin; NC, negative control; si, small interfering RNA.
