KT-extrinsic activity of Mps1 contributes to Mad1 KT recruitment. (A and B) Immunofluorescence images (A) and quantifications (B) of Mad1 and Mps1 levels at unattached KTs of neuroblasts from w¹¹¹⁸ or homozygous mps1^G4422 flies. When indicated, EGFP-Mps1-C^term or EGFP-Mps1-WT transgenes were expressed under control of Mps1 native promoter in a homozygous mps1^G4422 background. To generate unattached KTs, neuroblasts were incubated with colchicine (50 µM) for 90 min. Mad1 and Mps1 fluorescence intensities were determined relative to Spc105 signal (n ≥ 106 KTs). (C) Western blot analysis of endogenous Mps1, EGFP-Mps1-WT, and EGFP-Mps1-C^term levels in total lysates of third instar larval brains from A. (D) Western blot analysis of Mps1, Megator, and Mad1 relative levels in control S2 cells and in cells depleted of the indicated proteins. Cells were incubated with MG123 (20 µM) for 1 h and with colchicine (30 µM) for 2 h. Asterisk denotes bands resulting from unspecific anti-GFP blotting. (E and F) Immunofluorescence images (E) and quantifications (F) of Mad1 and Mps1 levels at unattached KTs of control S2 cells and cells depleted of the indicated proteins. Cells were incubated with MG123 (20 µM) for 3 h and with colchicine (30 µM) for 2 h. Mad1 and Mps1 fluorescence intensities were determined relative to CID signal (n ≥ 109 KT for Mad1, n ≥ 139 KTs for Mps1). (G) Mitotic index quantification based on H3^Ser10Ph staining of control S2 cells and cells depleted of the indicated proteins. Cells were incubated with colchicine (30 µM) for the indicated time periods. In B, F, and G, data are presented as mean ± SD. *, P < 0.05; ****, P < 0.0001 (Kruskal–Wallis, Dunn’s multiple comparison test). Scale bars, 5 µm (inset, 0.5 µm).