RNAi depletion of Endos or its targeting to the nucleus results in Tws-dependent mitotic defects (complements to Figs. 8 and 9). (A) Cells stably expressing H2A-RFP and lamin-GFP were transfected with dsRNA against Endos or the bacterial KAN gene (nontarget control). Cell divisions were then filmed on a spinning-disk microscope. Upon depletion of Endos, mitosis is delayed, and chromosomes become scattered and/or lagging. Lamin-GFP is ultimately recruited aberrantly on dispersed chromatin masses. Scale bar: 5 µm. (B) Quantification of the percentage of mitotic cells displaying scattered chromosomes (mean ± SD). (C) WB validation of protein expression and RNAi depletion for experiment in Fig. 8 A. (D and E) Independent validation with cells constitutively expressing Endos-Flag variants under the pAC5 promoter. (D) WB. (E) Quantification of phenotypes (mean ± SD, n = 3). *, P < 0.05; ****, P < 0.0001. (F) WB validation of protein expression and RNAi depletion for experiments in Fig. 9. chrom., chromosomes; end., endogenous; expo, exposure.