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. 2018 Oct 17;47(1):134–151. doi: 10.1093/nar/gky945

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© The Author(s) 2018. 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 (http://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 6. — The impact of MCM2 on cilia occurs also in non-replicating cells. (A) siRNA transfections were carried out after 1 day or serum starvation. Then, transcriptional changes indicative of cilia and centrosome impairment were monitored and cilia length was measured. (B) Human fibroblasts serum starved for 24 hours have ceased to express markers indicative of S-phase (Cyclin A, CCNA) or cycling cells (PCNA). Western blot of cells cultured in regular serum-containing medium (A) and of cells starved for 24 h. (C) Quantification of CCNA expression (normalized to βActin) in cells cultured either in full (A) or serum-free medium (S). n = 4. (D) Quantification of PCNA expression (normalized to βActin) in cells cultured either in full (A) or serum-free medium (S). n = 4. (E) MCM2 siRNA transfection in cells serum-starved for 24 hours significantly upregulates genes implicated in cilia shortening and centriole duplication. Expression levels were normalized to levels in siCTRL transfected cells (indicated by dashed line intersecting the y axis at 1). n = 4, paired, two-tailed t-test with Welch's correction. Mcm2: P < 0.0001; Ube2c: P = 0.0357; Prc1: P = 0.0123; Cdc25c: P = 0.0134; Nek2: P = 0.0322; Aurka: P = 0.0110; Poc1a: P < 0.0001; Stil: P = 0.0133; Plk1: P = 0.0437; Plk4: P = 0.0246. (F) MCM2 knockdown in non-replicating cells shortens cilia. Scale bar: 5 μm. (G) Primary cilia form at similar rates upon MCM2 knockdown in non-replicating cells. P = 0.8957, unpaired, two-tailed t-test with Welch's correction. Number of cells: siCTRL = 138 cells; siMCM2 = 146 cells (n = 3 experiments). (H) Primary cilia are shorter upon MCM2 depletion in G0. P < 0.0001, unpaired, two-tailed t-test with Welch's correction. 3 experiments. n = 71 cilia (siCTRL) and 69 cilia (siMCM2) from three transfections.