Figure 6.

NOL7 siRNA depletion reduces global protein synthesis and causes the nucleolar stress response in MCF10A cells.

A. NOL7 siRNA depletion reduces global protein synthesis. MCF10A cells were depleted with siRNAs for 72 h, followed by a 1 h treatment of 1 µM puromycin. Total protein was analysed by western blot. Representative western blot shown where Mock and siNT are negative controls, siNOL11 is a positive control. Mock 0.5 µM was included to confirm robustness of the signal quantification. β-actin was used as a loading control.

B. Quantification of the decrease in global protein synthesis after NOL7 siRNA depletion in MCF10A cells. Quantification of western blots from (A) normalized to β-actin signal and relative to siNT negative control. 3 biological replicates, plotted mean ± SD. Data were analysed by one-way ANOVA with Dunnett’s multiple comparisons test, ** p ≤ 0.01.

C-E. siNOL7 depletion induces the nucleolar stress response in MCF10A cells.

C. NOL7 siRNA depletion leads to increased TP53 protein levels in MCF10A cells. Representative western blot for TP53 shown where Mock and siNT are negative controls, siNOL11 is a positive control. β-actin is a loading control.

D. Quantification of the increase in TP53 levels after NOL7 siRNA depletion in MCF10A cells. Quantification of western blots from (C) normalized to β-actin signal and relative to siNT negative control. 5 biological replicates, plotted mean ± SD. Data were analysed by one-way ANOVA with Dunnett’s multiple comparisons test, * p ≤ 0.05.

E. NOL7 siRNA depletion significantly increases CDKN1A (p21) mRNA levels in MCF10A cells. qRT-PCR measuring CDKN1A mRNA levels. 2^{− ΔΔCt} were measured relative to 7SL internal control and siNT negative control sample. siNOL11 is a positive control. 3 technical replicates of 3 biological replicates, plotted mean ± SD. Data were analysed by one-way ANOVA with Dunnett’s multiple comparisons test, *** p ≤ 0.001, * p ≤ 0.05.