Fig 2: C23 expression levels were elevated in cancer cells and critical for cancer cell proliferationA. Protein level of C23 was determined by western blot in different cancer cell lines. GAPDH was used as loading control. B-C. C23 expression levels in human cancer lesions and corresponding normal adjacent tissues were analyzed by Western blot analysis (B) and Graphpad prism (C). D. C23 protein levels were evaluated by western blot in HCT116 cells stably expressing the control shRNA, C23 shRNA1 or C23 shRNA2. GAPDH served as loading control. E. The number of HCT116 cells stably expressing the control shRNA, C23 shRNA1 or C23 shRNA2 was determined by cell counter. The data were represented as mean±S.D. of three independent experiments. F. HCT116 cells stably expressing the control shRNA, C23 shRNA1 or C23 shRNA2 were treated with 100ng/ml Doxorubicin for 24 h. Viability of cells was determined using MTT assays by measuring the absorbance at 490 nm in a microplate reader. G. Long-term colony formation assay of HCT116 cells with and without stable knockdown of C23. Cells (5000 cells per well) were allowed to grow for 3 weeks, then stained and photographed. Scale bar, 1cm.

Fig 3: Schematic summarizing key observations of the current study.NIC4 mobility in the nucleus and nucleolus is dynamic with NIC4 localization to the nucleolus, guided by its NoLS. NIC4 associates with NCL and NPM, and protection from genomic stressors is dependent on nucleolar proteins NCL and FBL. Nucleolar functions can be uncoupled from nuclear activities of NIC4. Not to scale.

Fig 4: C23 suppressed the transcriptional activity of p53 upon DNA damage and hypoxiaA. pGL3-3×p53-BS-LUC and Renilla were co-transfected in HCT116 cells combined with and without C23 shRNA. 24 hours after transfection, cells were treated with Doxorubicin (100ng/ml) or upon 1% O2 for indicated times and the cell lysates were analyzed by lucifease assay. B. HCT116 cells with and without stable overexpressing C23 were co-transfected with pGL3-3×p53-BS-LUC and Renilla. 24 hours after transfection, cells were treated with Doxorubicin (100ng/ml) or upon 1% O2 for indicated times and then the cell lysates were subjected to lucifease assays. C. HCT116 cells with and without stable knockdown of C23 were treated with Doxorubicin (100ng/ml) for 24 hours, then p53 target gene mRNA expression levels were analyzed by qRT-PCR analysis. D-E. HCT116 cells with and without stable overexpressing C23 (a) or stable knockdown of C23(b) were treated with Doxorubicin (100ng/ml) or mock control for 24 hours. Cell lysates were then subjected to Western blot analysis with the indicated antibodies (D) and caspase3/7 activity analysis (E), respectively. F. (a) HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA. (b) HCT116 cells with and without p53 knockdown were introduced with C23 respectively. Protein level of C23 and p53 were evaluated by Western blot. GAPDH served as loading control. G. HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA (a) and HCT116 cells stably knocked down p53 were additionally introduced with C23 respectively (b). Cell number was evaluated by cell counter. The data were represented as mean±S.D. from three independent experiments. H. (a) HCT116 cells stably expressing the control shRNA, C23 shRNA, p53 shRNA,or C23 shRNA plus p53 shRNA. (b) HCT116 cells stably knocked down p53 were co-transfected with C23 cDNA respectively. Cells were then treated with 100ng/ml Doxorubicin for 24 hours. Cell viability was determined by MTT assay. The data were shown as the mean±s.d. of three independent experiments.

Fig 5: Upregulation of NCL promotes SVV replication. (A) Fluorescence of packed lentiviruses in HEK-293FT cells, and lentivirus-transduced BHK-21 cells expressing GFP, NCL-GFP, and FBL-GFP were captured with fluorescence microscopy at 24 h posttransfection and transduction. (B) Cell viability of lentivirus transduced BHK-21 cells were determined by CCK-8 assay (NS, not significant). (C to D) BHK-21 cells were transduced with lentivirus expressing GFP, NCL-GFP, and FBL-GFP. Cells were infected with SVV (MOI = 5) after 48 h postransduction, and the expression of VP1 was analyzed by TCID50 assay and Western blotting with antibodies to GFP, VP1, and β-actin at 0, 6, and 12 hpi, respectively. Statistical analysis was performed using GraphPad Prism. The error bars indicated mean ± SD from three independent experiments (*, P < 0.05; ***, P < 0.001; NS, not significant). (E) The expression of VP1 was quantified normalized against β-actin using Image J. Statistical analysis was performed using GraphPad Prism. The error bars indicated mean ± SD from three independent experiments (*, P < 0.05; ***, P < 0.001; NS, not significant).