Figure 7. GTP abundance regulates Pol I function in Mychi cells in part through GPN1 and GPN3.
(A and B) Immunoprecipitation with anti-RPA1 or rabbit IgG followed by Western blot for RPA1, GPN3-Myc, or GPN1-Myc in H82 cells with CRISPR/Cas9-mediated knockout of GPN3 or GPN1 followed by reexpression of wild-type or mutant GPN3 or GPN1. (C) Abundance of native GPN3 and Myc-tagged GPN3 in DMS53-CR cells with CRISPR/Cas9-mediated GPN3 knockout and reexpression of empty vector (EV), wild-type, or mutant GPN3. KO, cells functionally null for GPN3; NT, nontargeting guide RNA. (D and E) Nuclear RPA1 immunofluorescence and sample images of cells from C. ****P < 0.0001. Original magnification, 63×. (F) qPCR for rDNA promoter and IGS sequences after ChIP with anti-RPA1 antibody or IgG control in cells from C. Data are ChIP enrichment with anti-RPA1 relative to IgG control. Data are shown as mean and SD (F). Statistical significance was assessed using 1-way ANOVA with Tukey’s multiple-comparison test (D and F). All experiments were repeated twice or more.