Fig 2: BAMBI inhibits osteosarcoma growth and invasion via the TGF-β-induced EMT signaling pathway. (A) BAMBI treatment suppressed TGF-β protein expression in SAOS2 and MG63 cells. (B) BAMBI treatment inhibited EMT markers E-cadherin, vimentin and Twist expression in SAOS2 and MG63 cells. (C) BAMBI treatment inhibited the expression and phosphorylation levels of Smad2 and Smad3 in SAOS2 and MG63 cells. (D) TGF-β receptor using SB431542 suppressed the expression and phosphorylation levels of Smad2 and Smad3 in SAOS2 and MG63 cells. (E) pTGF-β antagonized the downregulated expression and phosphorylation of Smad2 and Smad3 in SAOS2 and MG63 cells. (F) pTGF-β increased EMT markers expression in BAMBI-treated SAOS2 and MG63 cells. pTGF-β inhibited the BAMBI-inhibited (G) growth and (H) invasion of SAOS2 and MG63 cells (magnification, ×200). pTGF-β, TGF-β overexpression; E-cadherin, epithelial-cadherin; BAMBI, bone morphogenetic protein and activin membrane-bound inhibitor; TGF-β, transforming growth factor-β; EMT, epithelial-to-mesenchymal transition. **P<0.01 vs. control group.

Fig 3: Analysis of BAMBI expression in osteosarcoma cell lines and clinical tumor tissues. Analysis of (A) mRNA and (B) protein expression levels of BAMBI in SAOS2 and MG63 cells, compared with normal cell line hFOB1.19. (C) BAMBI is downregulated in osteosarcoma tissues, compared with normal adjacent tissues (magnification, ×200). (D) Analysis of BAMBI expression and prognostic markers in osteosarcoma. **P<0.01 vs. control group. NS, no significant differences; BAMBI, bone morphogenetic protein and activin membrane-bound inhibitor.

Fig 4: BAMBI regulated the growth and aggressiveness of gastric cancer cells through the TGF-β/EMT signaling pathway. (A) Proliferation marker IPO-38 and Ki67 protein expression levels in BGC-823 cells following BAMBI overexpression were assessed. (B) Intracellular pH changes in BAMBI-overexpressed BGC-823 cells were analyzed. (C) EMT marker TWIST1, MMP9 and SOX4 protein expression levels in BGC-823 cells following transfection with BAMBI were indicated. (D) EMT transcription factor SNAI1, ACTA2 and VIM protein expression levels in BGC-823 cells following transfection with BAMBI were determined. Effects of BAMBI (E) overexpression or (F) knockdown on TGF-β and its regulatory molecules expression levels of N-cadherin, CT-I and FIB in BGC-823 cells. Effects of BAMBI (G) overexpression or (H) knockdown on TGF-β-induced EMT signal pathway in BGC-823 cells. One-way analysis of variance or two-tailed Student's t-test were performed. **P<0.01 as indicated. Control, pvector. BAMBI, bone morphogenetic protein and activin membrane-bound inhibitor; TGF-β, transforming growth factor-β; EMT, epithelial-mesenchymal transition; Si, small interfering RNA; pBAMBI, overexpressed BAMBI; TWIST1, twist-related protein 1; MMP9, matrix metallopeptidase; SOX4, SRY-box 4; ACTA2, α-actin-2; VIM, vimentin; FIB, fibronectin; CT-I; collagen-I.

Fig 5: In vivo efficacy of BAMBI reconstitution in xenografted mice. (A) BAMBI treatment significantly suppressed osteosarcoma growth. (B) BAMBI treatment significantly decreased osteosarcoma weight. (C) BAMBI treatment increased the number of apoptotic cells and decreased the TGF-β expression in tumor sections (magnification, ×200). (D) BAMBI treatment downregulated EMT markers E-cadherin, vimentin and Twist expression in tumor sections (magnification, ×200). (E) Evaluation of the median overall survival of xenografted mice. **P<0.01 vs. control group. E-cadherin, epithelial-cadherin; BAMBI, bone morphogenetic protein and activin membrane-bound inhibitor; TGF-β, transforming growth factor-β; EMT, epithelial-to-mesenchymal transition.