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. 2020 Jun 1;48(12):6583–6596. doi: 10.1093/nar/gkaa449

Figure 3.

Figure 3.

All components of the NWC complex are required for chromosomal association of this complex. (A) Effect of NOL11, WDR43, or Cirhin knockdown on the chromosomal association of the NWC complex. HeLa cells transfected with luciferase-specific siRNA (siCont.), NOL11-specific siRNA (siNOL11), WDR43-specific siRNA (siWDR43), or Cirhin-specific siRNA (siCirhin) were synchronised in mitosis and fractionated. Whole-cell extracts and chromosomal fractions were immunoblotted with the indicated antibodies. (B) Dissociation of NOL11 from the PR through knockdown of WDR43 or Cirhin. FLAG/HA-NOL11 HeLa cells transfected with siCont., siWDR43, or siCirhin were fixed with 4% PFA after pre-extraction and co-stained with anti-HA antibodies (NOL11; green) or anti-Ki67 antibodies (red) and then DAPI (blue). Representative cells in metaphase are shown. Scale bar, 2 μm. For each condition, the relative intensity of NOL11 and Ki67 signals at the PR was measured and shown in the graph. Values are mean ± SEM (n = 3), **P < 0.01, and ***P < 0.001, n.s. not statistically different (one-tailed Welch's t-test). Chromosomes from >25 mitotic cells were measured. (C) Translocation of the NOL11 from the nucleolus or mitotic chromosomes in the presence of RNase A. HeLa cells in interphase (top and second rows) and mitosis (third and bottom rows) were permeabilised and incubated without (Cont.) or with RNase A. Samples were subsequently fixed and stained with anti-NOL11 antibodies (red), anti-UBF antibodies (green), and DAPI (blue). Scale bar, 5 μm. (D) Association between pre-RNA and the NWC complex. The NWC complex was immunoprecipitated from mitotic HeLa cells that stably expressed FLAG/HA-NOL11 using anti-FLAG antibodies, and associated RNA was analysed using quantitative RT-PCR using specific primer sets for pre-rRNA (5′ETS regions) or β-actin mRNA. Values are mean ± SEM (n = 3). *P < 0.05, n.s. not statistically different (one-tailed Welch's t-test). (E) RNA-binding activity of Cirhin. RNA purified from HeLa cells was incubated with glutathione Sepharose-immobilised GST (lane 1) or GST fusion proteins of each component of the NWC complex (lanes 2–4). After extensive washing, bound RNA was analysed using quantitative RT-PCR using specific primer sets for pre-rRNA (5′ETS regions). Results were normalized to those for GST. Values are mean ± SEM (n = 3). *P < 0.05, **P < 0.01, n.s. not statistically different (one-tailed Welch's t-test).