Complete loss of Zwint1, RZZ, and Mad1 at Knl1-depleted kinetochores. (A–I) Nocodazole-treated HeLa cells treated with either control luciferase or Knl1 siRNA were immunostained using anti-Hec1 9G3 (A, C, E, and G) or anti-Knl1 (H) and either anti-Knl1 (A), anti-Zwint1 (B), anti-Rod (E), anti-Zwilch (G), or anti-Mad1 (H) antibodies. Kinetochore fluorescence was normalized relative to Hec1 (B, D, and F) or ACA (I) in control and Knl1-depleted cells. n = 100 kinetochores; P < 0.001. Error bars are SD from the means. (J) Images showing kinetochore localization of GFP-Zw10 in metaphase one-cell-stage embryos of control and kbp-5Δ (ok1358) alleles expressing mCherry-H2B. Bars, 5 µm.