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. 2022 Sep 26;50(18):10695–10716. doi: 10.1093/nar/gkac817

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 9. — Depletion of NOP2/NSUN1 reduces SNORD3/U3 and SNORD118/U8 co-sedimentation with 47S rRNA-containing pre-ribosomal particles. (A) HCT116 cells expressing doxycycline-inducible NOP2 shRNA were induced with 200 ng/ml doxycycline (Dox+) for 6 days. Non-induced (Dox–) cells were used as control. High molecular weight complexes containing pre-rRNAs and tightly associated ribosome assembly factors isolated from HCT116 nuclear extracts under high-salt conditions were separated on sucrose gradient ultra-centrifugation followed by fractionation. RNA was isolated from each fraction and analyzed by Northern blot using SNORD3/U3, SNORD118/U8 and 5′ETS probes. (B) Densitometry quantification of SNORD3/U3 and SNORD118/U8 signal from (A) normalized to the respective total signal from all fractions. Data are presented as the mean of three independent biological replicates ± standard deviation (SD). Statistical significance between NOP2 depleted samples (Dox+) and non-induced control samples (Dox–) was calculated using a two-tailed independent Student's t-test. (C) A small fraction of cells from (A) were collected to determine NOP2/NSUN1 depletion efficiency using western blot with the indicated antibodies.