Fig 1: CHRDL2 blocked BMP-9 and regulated PI3K/AKT pathway. (a) Immunoprecipitation demonstrated the binding between BMP-9, BMP-7, BMP-4, BMP-2, and CHRDL2. (b,c) Western blot was used to detect the expression of BMP-9 while CHRDL2 was overexpressed or inhibited. (d) Immunoprecipitation was performed to detect the combination between BMP-9 and ALK1 while CHRDL2 was inhibited. (e) Immumohistochemical staining was used to detect the expression relationship between CHRDL2 and BMP-9 in osteosarcoma tissues (magnification ×400). (f) GSEA analysis showed CHRDL2 had potential to regulate PI3K/AKT pathway according to the gene expression profile GSE87624 and GSE99671. (g) Western blot was used to detect the expression of PI3K, p-PI3K, AKT, p-AKT, and MMP-9 while CHRDL2 was inhibited. (h) Western blot was used to detect the expression of PI3K, p-PI3K, AKT, p-AKT, and MMP-9 while CHRDL2 was overexpressed. BMP-9, bone morphogenetic protein 9; CHRDL2, chordin-like 2; GSEA, gene set enrichment analysis
Fig 2: Overexpressed BMP-9 reversed the effects of CHRDL2 overexpression on regulating PI3K/AKT pathway, proliferation and mobility. (a) Western blot was used to detect the expression of PI3K, p-PI3K, AKT, p-AKT, and MMP-9 while BMP-9 was overexpressed in CHRDL2 overexpression cells. (b) CCK-8 was used to detect the proliferation while BMP-9 was overexpressed in CHRDL2 overexpression cells. (c) Colony formation assay was used to detect the colony formation while BMP-9 was overexpressed in CHRDL2 overexpression cells. (d,e) Wound healing assay and transwell assay were used to determine the mobility while BMP-9 was overexpressed in CHRDL2 overexpression cells. *p < .05; **p < .01. BMP-9, bone morphogenetic protein 9; CHRDL2, chordin-like 2; GSEA, gene set enrichment analysis
Fig 3: CHRDL2 was highly expressed in osteosarcoma tissues and cell lines. (a) qRT-PCR was used to detected the expression of CHRDL2 in osteosarcoma tissues (n = 25) and adjacent tissues (n = 25). (b) Immunohistochemistry was used to detect the expression of CHRDL2 in osteosarcoma tissues and adjacent tissues (magnification ×200. (c) qRT-PCR was used to detected the mRNA levels of CHRDL2 in normal osteoblast hFOB cells and osteosarcoma cell lines MG63, U2OS, Saos-2, and 143B. (d) Western blot was used to detected the protein levels of CHRDL2 in normal osteoblast hFOB cells and osteosarcoma cell lines MG63, U2OS, Saos-2, and 143B. *p < .05; **p < .01. CHRDL2, chordin-like 2; mRNA, messenger RNA; qRT-PCR, quantitative reverse transcription polymerase chain reaction
Fig 4: Inhibition of CHRDL2 decreased osteosarcoma cell proliferation and mobility. (a) The efficiency of targeted CHRDL2 short hairpin RNA (sh-CHRDL2) was detected by PCR. (b) The efficiency of sh-CHRDL2 was detected by western blot. (c) CCK8 assays were performed to detect the effects of CHRDL2 inhibition on osteosarcoma cell proliferation. (d) Colony formation assays were performed the he effect of CHRDL2 inhibition on osteosarcoma cell colony formation. (e) Wound healing assay was used to detect the effects of CHRDL2 inhibition on osteosarcoma cell migration. (f) Transwell assay was used to detect the effects of CHRDL2 inhibition on osteosarcoma cell invasion. *p < .05; **p < .01. CCK-8, cell counting kit-8; CHRDL2, chordin-like 2; PCR, qRT-PCR, polymerase chain reaction
Fig 5: Overexpression of CHRDL2 promoted osteosarcoma cell proliferation and mobility. (a) The efficiency of CHRDL2 overexpressed lentivirus (lv-CHRDL2) was detected by PCR. (b) The efficiency of lv-CHRDL2 was detected by western blot. (c) CCK8 assays were performed to detect the effects of CHRDL2 overexpression on osteosarcoma cell proliferation. (d) Colony formation assays were performed to detect the effects of CHRDL2 overexpression on osteosarcoma cell colony formation. (e) Wound healing assay was used to detect the effects of CHRDL2 overexpression on osteosarcoma cell migration. (f) Transwell assay was used to detect the effects of CHRDL2 overexpression on osteosarcoma cell invasion. *p < .05; **p < .01. CCK-8, cell counting kit-8; CHRDL2, chordin-like 2; PCR, qRT-PCR, polymerase chain reaction
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