Figure 4

Consequences of limiting CHD1L levels by siRNA. (A). Careful titration of CHD1L siRNA conditions were undertaken in A549 so as to mimic haploinsufficient expression of CHD1L. Left panels:These show the Western analysis of whole cell extracts from Untreated (Unt; mock-treated) control whereas siRNA indicates cells treated with CHD1L siRNA. β-tubulin expression was monitored to confirm equal loading. Right graph: Densiometric quantification of CHD1L expression, normalized to β-tubulin loading from three separate siRNA experiments. The degree of CHD1L reduction is very similar to that observed from the 1q21.1 deletion (Del) containing LBC (Fig 3A and B). Data represents the mean ± s.d. of three separate experiments (a.u. arbitrary units). (B). Inset image shows a typical catenated pseudomitotic cell following CHD1L siRNA-mediated knockdown in A549 treated with Topo II inhibitor (ICRF-193). The % pseudomitosis were enumerated under various conditions in A549 following CHD1L siRNA-mediated knockdown to mimic haploinsufficiency. Unt (untreated; not treated with ICRF-193), ICRF-193 (treated with ICRF-193), Con (control siRNA scrambled oligo), CHD1L siRNA (treated with siRNA to mimic CHD1L haploinsufficiency). Data represents the mean ± s.d. of three separate experiments and p < 0.005 for increase in Pseudomitosis (%) following CHD1L siRNA. (C). Inset image shows micronuclei (MN). The % of ICRF-193-induced MN in binucleate cells were determined in wild type (WT), Dup and Del containing LBLs following a 24 hr recovery from an overnight treatment with ICRF-193. Data represents the mean ± s.d. of three separate experiments and p = 0.02 for increase % MN in binucleates for Dup and p < 0.005 for Del containing LBCs.