Fig. 4: Dbx1 is the most overexpressed gene in ASP^cKO tumors and has a functional role in human FN-RMS.

a Gene expression by real-time PCR of Dbx1 (P = 0.0001) in ASP^WT tumors (n = 4) ASP^cKO tumors (n = 4), in duplicate. b DBX1 mRNA expression (log₂(TPM + 1)) in cell lines from the Broad Institute Cancer Dependency Map (DepMap), 20Q3 release. Soft-tissue sarcoma cell lines are in red. The number of cell lines is indicated along the x axis label for each respective tumor type. c DBX1 expression by RNA sequencing (FPKM) in human FN-RMS (n = 64), FP-RMS (PAX3-FOXO1 (P3F, n = 29) and PAX7-FOXO1 (P7F, n = 10)) tumors, and human skeletal muscle samples and human myoblast tissue cultures (n = 26) (SkM) from the NCI³⁰. d Gene expression by real-time PCR of DBX1 in RMS PDXs and human skeletal muscle myoblasts (HSMMs), in triplicate. e Gene expression by real-time PCR of DBX1 in SJRHB015721_X1 cell line following lentiviral transduction (72 h) with DBX1-targeting or control shRNA (Scr), in triplicate. (Scr-shDBX1-A P = 0.0068, Scr-shDBX1-B P = 0.0039). f Cell viability by Cell Titer Glo assay of SJRHB015721_X1-derived adherent cell cultures transduced with DBX1-targeting or control shRNAs after 5 days; in quadruplicate, compared to scramble. (Scr-shDBX1-A P < 0.0001, Scr-shDBX1-B P < 0.0001). g Volcano plot depicting differentially expressed genes between two DBX1-targeted and control shRNAs (absolute log fold change >0.58, −log₁₀(adjusted P < 0.5) in SJRHB015721_X1 cells. h, i GO analyses of genes with decreased (h) and increased (i) expression following shRNA-mediated DBX1 depletion in the SJRHB015721_X1 cell line. Top ten unique GO terms are shown, DAVID analysis. j, k Tumor volume of SJRHB015721_X1 orthotopic patient-derived xenograft (PDX) tumor cells transduced with DBX1-targeting or control shRNAs (j, P < 0.0001) and tumor mass (k, P < 0.0001), n = 10. l Representative H&E staining and IHC for DESMIN, MYOD1, and GFP from tumors in (j). Scale bar = 25 μm. All P values in pairwise comparisons were determined by Student’s t test (unpaired, two-tailed); **P < 0.01, ****P < 0.0001. Data represented as mean + SEM. Real-time PCR normalized to 18S rRNA and expressed relative to ASP^WT (a), the average of the HSMM cell lines (d), or scramble (e). See Supplementary Fig. 6 and Supplementary Data 1 and  2. Source data are provided as a Source Data file.