Fig 2: Effects of RALA overexpression on growth of OS xenografts in mice. (a–c) Tumors harvested from male SCID mice (nu/nu; age, 6 weeks; weight, 18 g) were subcutaneously injected into RALA-overexpressed or control MG63 cells (2 × 106 cells/mouse, six mice/group). Photos of tumors (a), tumor volume (b), and tumor weight (c) were presented. *p < 0.05. (d–f) H&E staining (d) was performed on xenografted tumors. Protein expression levels of RALA and Ki67 were detected using immunohistochemical assay (d) and quantitatively analyzed in (e) and (f). **p < 0.01; ****p < 0.0001. (g) Protein expression levels of RALA in OS xenografted tumors were detected using Western blotting.

Fig 3: Immune infiltration analysis and DNA methylation analysis of RALA in OS. (a, b) Enrichment scores (a) of RALA in different immune cells in sarcoma. The positive correlation and significance were given in the heat map (b). **p < 0.01; ***p < 0.001. (c) Correlation between RALA and immune cells was presented using TIMER tool. (d, e) Single-cell sequencing results show differential expression of RALA in immune cells and stromal cells (d). Expression levels of RALA in different immune cells were given in (e). **p < 0.01. (f) Correlation between RALA expression and DNA methylation level was detected in different sites of RALA TSS in OS. (g–j) Correlation between m6A-related genes and RALA in OS tissues from TARGET was presented using coexpression heat map, containing writers (g), reader (h), and erasers (i). Positive correlation and significance were given in the heat map (j). *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4: Molecular network and function of RALA in OS. (a) Molecular interaction network of RALA was analyzed using the STRING tool. (b–e) TOP 5 coexpression genes in different tumors were downloaded from GEPIA2. Correlation between the 5 genes; RALA and ABCE1 expression were presented in sarcoma tissues of TCGA using coexpression heat map (b) and chord diagram (d). Data of OS in TARGET was presented in (c) and (e). *p < 0.05; **p < 0.01; ***p < 0.001. (f) An intersection analysis of the SND1-binding and correlated genes was conducted. (g, h) Coexpression degree between RALA and ABCE1 in TCGA (g) and TARGET (h) dataset. (i–m) KEGG/GO enrich analyses were performed based on the 50 RALA-binding and 100 interacted genes, containing MF (i), BP (j), CC (k), KEGG pathway (l), and KEGG/GO network (m). (n–s) Functional experiments were performed in MG63 cells. Proliferative ability was detected using EDU assay with representative images (n) and quantitative analysis (q); nuclei stained with DAPI (blue); proliferating cells stained with EDU (red). Migratory ability was detected using wound healing assay with representative images (o) and quantitative analysis (p). Invasive ability was detected using invasion assay with representative images (s) and quantitative analysis (r); cells that pass through Matrigel are stained with 0.2% purple crystal (purple). **p < 0.01.

Fig 5: Protein and mRNA expression levels of RALA in OS. (a) mRNA expression level of RALA in OS was analyzed using GEPIA2. (b–f) Survival probability of OS patients in high- and low-RALA groups in TCGA and TARGET database using Kaplan-Meier analysis. (b–d) Overall survival, disease-specific survival, and progress-free survival in TCGA. (e, f) Overall survival and disease-free survival in TARGET. (g, h) RNA-sequencing analyses showed differentially expressed genes, using volcano plots (g) and heat map (h). (i, j) RALA expression status was detected in fresh OS tissues and paired normal specimens. Protein expression level was detected using Western blotting. (i) mRNA expression level was detected using RT-qPCR. ****p < 0.0001. mRNA expression level in osteoblast (HFOB1.19) and OS cell lines (U2OS, 143B, and MG63) was detected using RT-qPCR. ***p < 0.001.