Figure 2.
BCL9L Knockdown Confers Aneuploidy Tolerance in HCT-116 Cells
(A) siRNA knockdown of BCL9L protein (top) and mRNA (bottom) in HCT-116 (representative experiments shown, Qiagen siRNA pool).
(B–D) Impact of 250 nM reversine treatment on cell viability (B), cell number (C), and fraction of BrdU-incorporating cells (D) following control or BCL9L siRNA transfection (72 hr, n = 3; p values calculated by paired Student's t test).
(E) Colony-forming efficiency of HCT-116 after siRNA transfection and 250 nM reversine treatment.
(F) Fold change induction of Caspase-3/7 enzymatic activity following 250 nM reversine treatment in HCT-116 transfected control siRNA or after BCL9L knockdown (72 hr, n = 3, p value calculated by paired Student's t test).
(G) Live-cell imaging analysis of post-mitotic daughter cell fate after a normal mitosis or mitosis with endogenous segregation errors. Daughter cells were tracked for at least 48 hr. Arrest was defined as the absence of cell division within 48 hr post mitosis. Death was defined as visible nuclear collapse.
(H) Effect of stable lentiviral BCL9L knockdown on colony-forming efficiency of HCT-116 treated with 125 nM reversine for 15 days and recovered for additional 15 days.
(I) Modal centromeric deviation (%) in the number of FISH signals (CEP 1, 2, 8, and 15) in reversine-treated and untreated HCT-116 cells with lentiviral stable BCL9L knockdown (n = 3, 400–500 nuclei scored per experiment). Cells were treated as in (H).
(J) Karyotypic analysis following stable lentiviral BCL9L knockdown and reversine treatment. Metaphases were stained with a pan-centromeric probe (70–100 cells analyzed, modal number of chromosomes and percentage deviating from the mode shown at the top; p values calculated by two-sided Wilcoxon rank-sum test).
Error bars denote SD. ns, not significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figures S4 and S5.