PAX3-FOXO1 suppresses TGFβ signaling and cell–ECM interaction. (a) Higher expression of genes enhanced by PAX3-FOXO1 knockdown (PF^KD) in FNRMS-like Subgroups 4 and 5, than in FPRMS-like Subgroup 3. The color indicates the average cellular expression level of genes highly expressed in PF^KD FPRMS cells compared with control cells. (b) GSEA indicates that genes highly expressed in Subgroup 4, compared with other subgroups, show statistically significant positive enrichment of genes upregulated by PF^KD in FPRMS cells. (c) GO analysis of highly expressed genes in PF^KD FPRMS cells (Rh30 and Rh41) compared with control cells, using GO Function gene sets as the reference gene set. (d and e) Immunoblotting and (e) its quantification show higher pMLC of PF^KD FPRMS cells than control cells, implying their higher cell–ECM interaction (n = 3). (f) IF images of control and PF^KD Rh30 and Rh41, stained for F-actin (with phalloidin) and vinculin, showing focal adhesion in PF^KD cells. (g) GO analysis of highly expressed genes in PF^KD FPRMS cells (Rh30 and Rh41) compared with control cells, using KEGG gene sets as the reference gene set. In c and g, color represents the count of inquired genes overlapping with each specific reference gene set. Circle size indicates adjusted P value, and strength means the ratio of inquired to reference gene counts, implying the enhanced expression of the corresponding reference. (h) Dynamic SMAD3 nuclear localization upon TGFβ treatment (4 ng/ml) in PF^KD FPRMS cell lines, monitored using NG-Smad3 live-cell sensor. (i) TGFβ signaling activity, quantified by the ratio of intensity in nuclear SMAD3 after TGFβ treatment to before the treatment, in PF^KD FPRMS cells. Each dot represents the value of a cell, and lines are averages of each cell line (minimum n = 8). (j) Immunoblotting of nuclear and cytoplasmic fractionized proteins for SMAD2/3, lamin B1 (housekeeping control for nuclear proteins), and GAPDH (housekeeping control for cytoplasmic proteins). SMAD2/3, lamin B1, and GAPDH blots are the same as in Fig. S3 c. (k) Quantification of nuclear proteins exhibits higher SMAD3 nuclear localization of PF^KD FPRMS cells than control cells, implying their higher TGFβ signaling activity (n = 3). In e, i, and k, error bars represent SE and statistical significance was assessed using two-sided Student’s t test or Wilcoxon’s rank sum test (i): *P < 0.05, **P < 0.01, and ***P < 0.005. Source data are available for this figure: SourceData F4.