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Figure 3 | Orphanet Journal of Rare Diseases

Figure 3

From: Understanding the impact of 1q21.1 copy number variant

Figure 3

Functional assays for CHD1L in patient cells. (A) Left panels: Western analysis of CHD1L expression from wild-type (WT), 1q21.1 deletion (Del) and duplication (Dup) LBCs following titration of whole cell extracts. Right Panels: β-tubulin re-probe to confirm equal loading. (B). Densiometric quantification of CHD1L expression from Western analysis from low (black bar), intermediate (white) to higher (grey) amounts of protein, from each line, using three separate determinations, normalized to β-tubulin loading (a.u. arbitrary units). p = 0.009 for Del and p < 0.005 for Dup compared to WT. (C). The Decatenation Checkpoint (DCC). Unreplicated DNA sequences between converging replication forks undergo catenation and torsional tension which is normally relieved by Topoisomerase IIα (Topo IIα) which induces a transient DSB enabling decatenation (untangling). DCC activation in G2 prevents entry into mitosis until sister chromatids are fully separated. DCC can be activated by Topo II inhibitors arresting the cycle in G2 (indicated in red). DCC failure is monitored by the enumeration of 'pseudomitosis' containing highly catenated (entangled) chromatids. Inset image shows typical pseudomitotic cells following treatment of the Del LBCs with the Topo II inhibitor, ICRF-193. (D). Mitotic index (Mitosis %) and Pseudomitotic index (Pseudomitosis %) for untreated (Unt) LBCs or ICRF-193 treated, for wild-type (WT), Werner's syndrome (WRN), Dup and Del containing LBCs. WRN LBCs are known to be defective in DCC activation. Data presented indicates the mean ± s.d of three separate determinations. p < 0.005 for reduction in Mitosis (%) Unt compared to ICRF-193 and p < 0.005 for increase in Pseudomitosis (%) Unt compared to ICRF-193.

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