Fig. 4

(A) Volcano plot of log₂ fold change (log2FC) gene expression (SIX1 KD over Scramble) and adjusted p value after differential expression analysis from SMS-CTR RNA-seq. Red and blue dots denote genes significantly upregulated and downregulated upon SIX1 KD, respectively.

(B) GSEA plots of ranked log2FC expression (SIX1 KD over Scramble) show positive enrichment for curated muscle cell differentiation and skeletal muscle contraction gene signatures and negative enrichment for chromatin assembly gene signatures.

(C) Heatmap expression of the Molecular Signatures DataBase (MSigDB) myogenesis gene set across Scramble and SIX1 KD samples. Scale bar represents Z score-converted log2CPM values.

(D) qRT-PCR of genes involved in muscle differentiation in SMS-CTR and RD cell lines with SIX1 KD. Each dot represents one independent biological replicate. Statistical differences were calculated using one-way ANOVA followed by post hoc Dunnett’s multiple comparisons test.

(E) MyHC (magenta) immunostaining and DAPI counterstain (yellow) in SIX1 KD RMS cells compared with Scramble RMS cells.

(F) Quantification of myHC staining over total nuclei per field of view; each dot represents the percentage of myHC+ cells over one technical replicate from at least 3 independent experiments.

(G)Fusion indices of SMS-CTR and RD control and SIX1 KD cells. Statistical differences were calculated by one-way ANOVA followed by Dunnett’s multiple comparisons post hoc test.