Reduced B7-H3 expression by PAX3-FOXO1 knockdown inhibits cellular motility and promotes myogenic differentiation in alveolar rhabdomyosarcoma

Figure 1. Knockdown of PAX3-FOXO1 downregulates the expression of B7-H3. (a) Quantitative real-time PCR (qRT-PCR) analysis of PAX3-FOXO1. Knockdown efficacy of PAX3-FOXO1 was confirmed in three PAX3-FOXO1 positive ARMS cell lines. RD, a PAX3-FOXO1-negative cell line, was used as a negative control. (b) qRT-PCR analysis of B7-H3. (c) Representative histogram of flow cytometry analysis of B7-H3. (d) Mean fluorescence index of B7-H3 in flow cytometry analysis. Results shown are means ± SD. *p < 0.05, **p < 0.01, n.s. p > 0.05, by two-tailed unpaired t test.

Figure 2. Gene expression analysis. Knockdown of PAX3-FOXO1 and B7-H3 reveal shared down/upregulated genes. (a) Venn diagram of genes that were downregulated by the knockdown of PAX3-FOXO1 or B7-H3. (b) Venn diagram of genes that were upregulated by the knockdown of PAX3-FOXO1 or B7-H3. (c) Heat map of the top 50 genes that were down/up-regulated by the knockdown of PAX3-FOXO1 or B7-H3. The left line shows Rh-30 siCont, the middle Rh-30 siPF, and the right siB7-H3.

Figure 3. Gene set enrichment analysis (GSEA). (a) Representative list of impoverished gene sets upon knockdown of PAX3-FOXO1 and B7-H3. (b) Enrichment plot from the GSEA. Interferon gamma production (left panel) and chemotaxis (right panel). (c) Representative list of enriched gene sets upon knockdown of PAX3-FOXO1 and B7-H3. (d) Enrichment plot from the GSEA. Actin filament polymerization (left panel) and actin binding (right panel).

Figure 4. Knockdown of PAX3-FOXO1 or B7-H3 attenuates cell migration. (a) Representative figure of a wound healing assay. (b) Relative healed wound area (% of original wound area) in a wound healing assay. Knockdown of PAX3-FOXO1 or B7-H3 attenuates cell migration. (c) Transwell assay. Relative luminescence units are shown as the quantification of migratory cells. Results shown are means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, by two-tailed unpaired t test.

Figure 5. CXCR4 and STYK1 expression levels are regulated by PAX3-FOXO1 and B7-H3. (a) Quantitative real-time PCR (qRT-PCR) analysis of CXCR4. (b) qRT-PCR analysis of STYK1. Results shown represent means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. p > 0.05, by two-tailed unpaired t test.

Figure 6. Knockdown of PAX3-FOXO1 and B7-H3 induces the expression of myogenin and may induce myoblast differentiation. (a) Quantitative real-time PCR analysis of myogenin. (b) Western blot analysis of myogenin. Knockdown of PAX3-FOXO1 (detected by anti-PAX3 antibody) and B7-H3 induces the expression of myogenin. (c) Immunofluorescence of myosin heavy chain. Results shown represent means ± SD. *p < 0.05, **p < 0.01, by two-tailed unpaired t test.