Panel A: A northern blot analysis and a line graph show the 30S slash 45S ratio in U2OS cells ectopically expressing either WDR75 wild-type or WDR75 p.T46I protein. The northern blot uses the ETS1-3636 probe to detect pre-rRNA precursors and mature rRNAs. The line graph quantifies the 30S slash 45S ratio, indicating a significant increase in the 30S slash 45S ratio in M1 cells compared to WT cells. Panel B: Another northern blot analysis and a line graph display the ratios of 41S slash 45S, 30S slash 41S, 21S slash 30S, and 18S-E slash 21S in the same cell types using the 5ITS1 probe. The line graph quantifies these ratios, showing variations between WT and M1 cells. Panel C: Two bar graphs illustrate the pre-rRNA product to precursor ratios for WT and M1 cells relative to EV controls. The plots show different ratios for various pre-rRNA species, with statistical significance indicated by asterisks. Panel D: Western blot images show the expression levels of WDR75-flag, p53, p21, and actin in U2OS cells with different treatments. Panel E: Three bar graphs quantify and statistically analyze the expression levels of WDR75-flag, p53, and p21 from Panel D, showing significant differences in expression levels between EV, WT, and M1 cells.
Altered pre-rRNA processing and increased p21 expression in U2OS cells with WDR75 inactivation and ectopic expression of WDR75 p.T46I. (A and B) Northern blot analysis of U2OS cells ectopically expressing a pWPI EV, WDR75 WT, or WDR75 p.T46I. The WDR75 gene locus was inactivated by CRISPR/Cas9 in WT- and WDR75 p.T46I (M1)-expressing cells. Indicated radiolabeled probes were used to detect pre-rRNA precursors and mature rRNAs. (A) ETS1-3636 probe, quantification of the 30S/45S ratio in WT and M1 cells. (B) 5′ITS1 probe, quantification of 41S/45S, 30S/41S, 21S/30S, and 18S-E/21S ratios in WT and M1 cells. (C) Pre-rRNA product-to-precursor ratios obtained for WT and M1 cells, relative to EV controls. Data represent mean values ± SEM from three independent experiments (n = 3). Statistical analysis presented in A–C was performed using paired Student’s t test; *P < 0.05; **P < 0.01. (D) Western blot analysis for p53, p21, and WDR75-Flag protein expression in the indicated U2OS cells. (E) Quantification and statistical analysis of WDR75-Flag, p53, and p21 expression levels from D. Statistical analysis was performed using one-way ANOVA. *P < 0.05; **P < 0.001. EV, empty vector. Source data are available for this figure: SourceData F4.