Figure 3.

Nucleolar protein NS and RPA194 is downregulated by QC treatment. (A) NS protein expression in various OC cell lines was assessed by western blot. αTubulin was used as loading control. (B) Kaplan–Meier progression-free survival (PFS) analysis shows high GNL3/NS expression is associated with worse PFS in OC patient’s cohort. (C) Relative expression changes for ribosomal subunits NS and RPA194 after 1 h of 1 µM QC treatment in OVCAR5 OC cells (normalized to RPLP0; n = 3) * (p < 0.05), ** (p < 0.01). (D) Immunoblot analysis of NS and RPA194 expression in OVCAR5 and (E) OVCAR8 cells upon QC treatment for 24 h in a dose dependent manner. α Tubulin was used as loading control. Densitometric analysis was performed using Image J, normalized to endogenous control and the fold change was provided beneath the panel. (F) shRNA-mediated stable NS knockdown OVCAR8 cells were generated, and the knockdown effect was validated by western blot analysis. αTubulin was used as loading control. Densitometric analysis was performed using Image J, normalized and fold change was plotted *** (p < 0.001). (G,H) Clonogenic OVCAR8 cell viability after NS knockdown compared to NTC transfection (n = 3). Error bars represent standard error, and significance is denoted by **** (p < 0.0001). (I) Western blot analysis was performed in untreated control and QC-treated HeyA8-MDR OC xenograft tumors against NS and RPA194 expressions. PCNA was used as loading control. Fold change was analyzed and provided beneath the panel. Abbreviations: QC: Quinacrine; NS/GLN3: Nucleostemin, RPA194: RNA Polymerase I Subunit A; NTC: non-targeted control, sh_NS: shRNA-mediated NS downregulation; PCNA: Proliferating cell nuclear antigen.