Fig 2: CDKAL1 promotes the assembly of the eIF4F translation initiation complex. A) Western blotting analysis of m7GTP precipitate from RD cell lysate. GAPDH, ß-actin, vinculin, and a-tubulin serve as negative controls. B) Western blotting analysis of m7GTP precipitate from RD cell lysate with urea-based denaturation. Numbers indicate the densitometry of signals (percent of control, Urea 0 m). C) An in situ proximity ligation assay using anti-myc tag antibody and antibodies against endogenous eIF4A, eIF4G, and eIF4E in parental RD cells and RD cells expressing myc-CDKAL1WT. Scale bars, 20 µm. D) An in situ proximity ligation assay using antibodies against endogenous proteins and m7G cap. Note that CD133high RD cells showed elevated expression levels of CDKAL1 compared to CD133low RD cells (Figure 1E). E) Western blotting analysis of m7GTP precipitate from Control-shRNA- or CDKAL1-shRNA-expressing RD cell lysate. F) Western blotting analysis of m7GTP precipitate from Control-shRNA-, CDKAL1-shRNA-, EIF4G-shRNA-, EIF4E-expressing RD cell lysate. G) A cartoon representing the mutants used in this study. H) Results of the rescue experiment with several truncated mutants of CDKAL1 to maintain the self-renewal capacity and SALL2 protein levels of RD cells (n = 4, error bars indicate mean ± SD). I) The m7GTP pull-down experiments with CDKAL1-shRNA-expressing RD cells displaying that the NH2-terminus of CDKAL1 is essential for sustaining the eIF4F complex formation.

Fig 3: CDKAL1 overexpression is associated with a worse prognosis in SALL2high RMS patients. A) SALL2 mRNA expression levels in normal skeletal muscle and fusion-negative and fusion-positive RMS in a gene set of GSE108022 are shown. B) Kaplan–Meier curve displaying the prognosis in RMS patients with SALL2high and SALL2low signature from the E-TABM-1202 dataset. C) Kaplan–Meier curve showing that elevated CDKAL1 expression levels worsen the prognosis of the SALL2high population of RMS patients. D) The expression levels of CDKAL1 and SALL2 in RMS patients at various clinical stages. E) Graphical summary of the roles of CDKAL1 in normal tissue and CSCs.

Fig 4: CDKAL1 promotes the maintenance of CSC-related traits in RMS. A) The self-renewal capacity of Control-shRNA- or CDKAL1-shRNA-expressing RMS cell lines was measured (n = 4, error bars indicate mean ± SD). Western blotting results are shown to confirm the CDKAL1 knockdown. B) The clonogenic growth potential of Control-shRNA- or CDKAL1-shRNA-expressing RMS cells (n = 3, error bars indicate mean ± SD). C) The cell proliferation analysis of Control-shRNA- or CDKAL1-shRNA-expressing RD and RH30 cells (n = 4, error bars indicate mean ± SD). D) Flow cytometry analysis showing the effect of CDKAL1 knockdown on CD133 expression in RD cells and RH30 cells. E) Immunofluorescent imaging of myogenic differentiation of RMS cells after CDKAL1 knockdown, as indicated by positive staining for myosin heavy chain (MyHC). F) One million Control-shRNA- or CDKAL1-shRNA-expressing RMS-YM cells were subcutaneously injected at the backs of BALB/c-nu/nu mice, and tumor growth was measured. Ten injection sites were measured for each group (error bars indicate mean ± SD). G) Immunofluorescent analysis using anti-CD133 and anti-MyHC antibodies in the RMS-YM xenografts obtained in Figure 2E. All scale bars, 50 µm. H) Effect of the forced expression of CDKAL1 on the sphere-forming capacity of RD and ICH-ERMS-1 cells (n = 4, error bars indicate mean ± SD).

Fig 5: Myoblastic C2C12 cells acquire the CSC-related properties that require CDKAL1 upon malignant transformation. A) Ectopic expression of HRas G12V and tp53-shRNA transforms C2C12 cells. B) Tumor-propagating capacity of transformed C2C12 cells. Note that the tumor consists of a mixed population of highly atypical undifferentiated and spindle-shaped cells with eosinophilic cytoplasm that were positive for desmin immunostaining. These findings are consistent with the pathology of embryonal RMS. Scale bars, 50 µm. C,D) Transformed C2C12 cells showed elevated expression levels of CDKAL1 and acquired the ability of anchorage-independent growth. Scale bars, 100 µm. E) HRas/shp53-C2C12 cells are insensitive to the differentiation cue, while Control-C2C12 cells form myotubes and express the myogenin protein, a master regulator of myodifferentiation. Scale bar, 100 µm. F,G) The self-renewal (F, n = 4, error bars indicate mean ± SD) and colony-forming (G, n = 3, error bars indicate mean ± SD) capacity of Control-shRNA- or Cdkal1-shRNA-expressing Control-C2C12 or HRas/shp53-C2C12 cells. H) Limiting dilution assay displaying the tumor-propagating capacity of HRas/shp53-C2C12 cells with Control-shRNA or Cdkal1-shRNA.