Fig. 3
Histone H3K9 and H4 acetylations facilitate initial CENP-AHCP−3 and NDC-80 deposition on newly formed ACs. a Immunofluorescence of CENP-AHCP−3 on first-generation ACs at different cell stages, and ACs that have been propagated for generations and endogenous chromosomes in GFP::LacI-, GFP::LacI::HDA-1- and GFP::LacI::HDA-1(H145A)-tethering strains. Cropped images containing ACs and endogenous chromosomes (Endo Chr.) were shown. Embryos were stained with antibody against CENP-AHCP−3 (red), antibody against LacI (green) and DAPI (blue), shown separately and merged. Scale bar represents 1 μm for both ACs and endogenous chromosomes. Examples of bipolar-oriented CENP-AHCP−3 are highlighted in red boxes. Quantification of IF signals. CENP-AHCP−3 signals were normalized with DAPI signals, and the average normalized CENP-AHCP−3 signal intensity was calculated. The number of cells (n) analyzed was indicated. Error bars indicate 95% confidence interval (CI) for the mean. ***p < 0.001 by Student’s t test. NS means not significant. Black arcs show comparisons between GFP::LacI-, GFP::LacI::HDA-1- and GFP::LacI::HDA-1(H145A)-tethering strain at the same cell stage. Blue arcs show comparisons between ACs at different stages in GFP::LacI-tethering strain. b Quantification of the % of cells with bipolar or diffused CENP-AHCP−3 on the newly formed ACs in 1 to 8-, 9 to 16- and 17 to 32-cell stage in GFP::LacI-, GFP::LacI::HDA-1- and GFP::LacI::HDA-1(H145A)-tethering strains. The number of cells (n) analyzed was indicated. *p < 0.05 by Chi-squared test. Black arcs show comparisons among GFP::LacI-, GFP::LacI::HDA-1- and GFP::LacI::HDA-1(H145A)-tethering strains at the same cell stage. Blue arcs show comparisons between ACs at different stages in GFP::LacI-tethering strain. Green arcs show comparisons between ACs at different stages in GFP::LacI::HDA-1(H145A)-tethering strain. c Immunofluorescence of NDC-80 on first-generation ACs at different cell stages, and ACs that have been propagated for generations and endogenous chromosomes in GFP::LacI- and GFP::LacI::HDA-1-tethering strains. Cropped images containing ACs and endogenous chromosomes (Endo Chr.) were shown. Embryos were stained with antibody against NDC-80 (red), antibody against LacI (green) and DAPI (blue), shown separately and merged. Scale bar represents 1 μm for both ACs and endogenous chromosomes. Quantification of IF signals. NDC-80 signals were normalized with DAPI signals, and the average normalized NDC-80 signal intensity was calculated. The number of cells (n) analyzed was indicated. Error bars indicate 95% confidence interval (CI) for the mean. ***p < 0.001 by Student’s t test. NS means not significant. Black arcs show comparisons between GFP::LacI- and GFP::LacI::HDA-1-tethering strains at the same cell stage. Blue arcs show comparisons between ACs at different stages in GFP::LacI-tethering strain