Fig 2: DDX21 and CEP55 play a role in neuroblastoma cell cytoskeletal stability. (A–B) DOX‐inducible control shRNA, DDX21 shRNA‐1, or DDX21 shRNA‐2 BE(2)‐C and Kelly cells were treated with vehicle control or DOX for 72 h, followed by immunostaining with an anti‐α‐tubulin antibody and counter‐staining of cell nuclei with DAPI (A). The areas covered by α‐tubulin filaments, relative to nucleus, were quantified (B). (C–D) BE(2)‐C and Kelly cells were transfected with control siRNA, CEP55 siRNA‐1, or CEP55 siRNA‐2 for 72 h followed by immunostaining with an anti‐α‐tubulin antibody and counter‐staining of cell nuclei with DAPI (C). The areas covered by α‐tubulin filaments, relative to nucleus, were quantified (D). Error bars represent standard error. * indicates P < 0.05, ** P < 0.01, and *** P < 0.001. Scale bar: 10 µm.

Fig 3: Schematic model of the regulation of DDX21 and CEP55 by N‐Myc. (A) N‐Myc binds to the DDX21 gene promoter containing the noncanonical E‐boxes, leading to DDX21 transcriptional activation, and increased DDX21 mRNA and protein expression. (B) DDX21 binds to the CEP55 gene promoter to facilitate CEP55 transcriptional elongation, leading to elevated CEP55 mRNA and protein expression, as well as cell cytoskeletal stability and cell proliferation. TSS, transcription start site.

Fig 4: DDX21 upregulates CEP55 gene expression by binding to the CEP55 gene promoter and promotes CEP55 transcriptional elongation. (A–B) DOX‐inducible control shRNA, DDX21 shRNA‐1, or DDX21 shRNA‐2 BE(2)‐C and Kelly cells were treated with vehicle control or DOX for 48 h, followed by RT‐PCR (A) or western blot (B) analyses of DDX21 and CEP55 mRNA and protein expression. (C) Schematic representation of the CEP55 gene promoter. TSS, transcription start site. (D–E) ChIP assays were performed with a control or anti‐DDX21 antibody (Ab), the immunoprecipitated protein was subjected to immunoblot analysis with an anti‐DDX21 antibody (D), and the immunoprecipitated DNA was examined by PCR (E) with primers targeting the negative control region (Amplicons A) or the CEP55 gene core promoter (Amplicons B) in BE(2)‐C cells. Fold enrichment of DDX21 at the CEP55 gene promoter was calculated as the difference in cycle thresholds obtained with the anti‐DDX21 Ab and with the control Ab. (F) DOX‐inducible control shRNA, DDX21 shRNA‐1, or DDX21 shRNA‐2 BE(2)‐C cells were transfected with empty vector control or CEP55 gene promoter (−884 to +165 bp) in LightSwitch reporter construct for 24 h, then treated with vehicle control or DOX for 30 h followed by luciferase assays. Luciferase activities were expressed as fold changes relative to control shRNA, DDX21 shRNA‐1, or DDX21 shRNA‐2 BE(2)‐C cells transfected with empty vector control and treated with vehicle control. (G–H) DOX‐inducible DDX21 shRNA‐1 BE(2)‐C cells were treated with vehicle control or DOX for 48 h, followed by ChIP assays with a control or an anti‐phos S2 Pol II antibody. The immunoprecipitated protein was subjected to immunoblot analysis with an anti‐phos S2 Pol II antibody (G), and the immunoprecipitated DNA was examined by PCR (H) with primers targeting the CEP55 promoter or the CEP55 3′‐UTR. Error bars represent standard error. * indicates P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: N‐Myc upregulates DDX21 gene expression by binding to the DDX21 gene promoter in neuroblastoma cells. (A–B) BE(2)‐C and Kelly cells were transfected with control siRNA, N‐Myc siRNA‐1, or N‐Myc siRNA‐2 for 48 h, followed by RT‐PCR (A) and western blot (B) analyses of N‐Myc and DDX21 mRNA and protein expression. (C–D) Tetracycline withdrawal‐inducible N‐Myc overexpression SHEP‐21N cells were treated with vehicle control or DOX for 48 h, followed by RT‐PCR (C) and western blot (D) analyses of N‐Myc and DDX21 mRNA and protein expression. (E) Schematic representation of the DDX21 gene promoter. TSS, transcription start site. (F–G) ChIP assays were performed with a control or an anti‐N‐Myc antibody (Ab), the immunoprecipitated protein was subjected to immunoblot analysis with an anti‐N‐Myc antibody (F), and the immunoprecipitated DNA was examined by PCR (G) with primers targeting a negative control region (Amplicon A) or the DDX21 gene promoter regions containing the E‐boxes (Amplicons B and C) in BE(2)‐C cells. Fold enrichment of N‐Myc at the DDX21 gene promoter regions was calculated as the difference in cycle thresholds obtained with the anti‐N‐Myc Ab and with the control Ab. (H) BE(2)‐C cells were transfected with control siRNA, N‐Myc siRNA‐1, or N‐Myc siRNA‐2 for 14 h, then transfected with empty vector control or DDX21 gene promoter (−870 to +111 bp) in LightSwitch reporter construct for 30 h followed by luciferase assays. Luciferase activities were expressed as fold changes relative to empty vector construct. Error bars represented standard error. * indicates P < 0.05, **P < 0.01, and ***P < 0.001.