(A) Polysomes analysis. H1944 and MCF-7 cells were depleted of U3 or U8 for two days and then treated with cycloheximide, to freeze the polysomes. Total extracts were analyzed by sucrose gradient centrifugation. The reduction of polysomes upon snoRNA depletion is obvious (red arrows). U3 depletion leads to a marked subunit imbalance due to a deficit in 40S, while U8 depletion affects primarily 60S accumulation. (B) Mature rRNA accumulation. H1944 and MCF-7 cells were depleted of U3 or U8, for 1, 2, or 3 days. Total RNA was extracted, resolved on denaturing gels, and mature rRNAs were visualized by ethidium bromide staining. 7SL, detected by Northern blotting, was used as a loading control. (C) Densitometric quantification of the signals shown in panel B. rRNA levels in cells depleted for U3 or U8 normalized with respect to the levels observed in cells treated with a non-targeting silencer (SCR). Note that after 72 h of U8 depletion, in addition to inhibition of 28S rRNA synthesis, the 18S and other rRNAs, were also reduced; this reflects the important reduction in cell viability observed at this time-point (Figure S2D).