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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 10. — The RNA methyltransferase activity of NOP2/NSUN1 is not required to maintain cell proliferation. (A) HCT116 WT or p53 –/– cells were transfected with non-targeting control (siC), NOP2 siRNA #1 or #2. 72h later, a fraction of the cells was harvested for western blot analysis with specific antibodies as indicated. (B) Densitometry quantification of each protein from (A) normalized to GAPDH. Data are presented as the mean of three independent biological replicates ± standard deviation (SD). Statistical significance between NOP2 depleted samples and non-targeting control samples was calculated using a two-tailed independent Student's t-test. A fraction of WT (C) and p53 –/– (D) HCT116 cells from (A) were monitored for proliferation daily using an MTT assay. Relative cell counts were normalized to non-targeting control (siC) on day 3. Data are presented as the mean of three independent biological replicates ± standard deviation (SD). Statistical significance between NOP2 depleted samples and non-targeting control samples was calculated using a two-tailed independent Student's t-test. *P < 0.05 and **P < 0.01: NOP2 siRNA#1 vs siC; ^#P < 0.05 and ^##P < 0.01: NOP2 siRNA#2 versus siC. (E) HCT116 cells expressing empty vector (Vector), siRNA resistant NOP2/NSUN1 WT or C513A mutant were transfected with non-targeting control (siC) or NOP2 siRNA #2. After 72 h, a fraction of cells was collected for western blot analysis with specific antibodies as indicated. (F) A small fraction of cells from (E) was monitored daily for proliferation using an MTT assay. Relative cell counts were normalized to empty vector with non-targeting control (siC + Vector) on day 3. Data are presented as the mean of three independent biological replicates ± standard deviation (SD). Statistical significance values relative to empty vector + NOP2/NSUN1 knockdown (siNOP2#2 + Vector) were calculated using two-tailed independent Student's t-test. *P < 0.05 and **P < 0.01: versus siC; ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001: versus siNOP2#2 + WT; ⁺P < 0.05, ⁺⁺P < 0.01 and ⁺⁺⁺P < 0.001: versus siNOP2#2 + C513A.