Fig. 5

RNA polymerase II-mediated transcription initiation is important for initial CENP-A^HCP−3 deposition on newly formed ACs. a Immunofluorescence of RNA polymerase II Serine 5 phosphorylation (RNA Pol II Ser5^P) on first-generation ACs at different cell stages, and endogenous chromosomes in GFP::LacI-tethering strain without and with alpha-amanitin treatment. Cropped images containing ACs and endogenous chromosomes (Endo Chr.) were shown. Embryos were stained with antibody against RNA Pol II Ser5^P (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. RNA Pol II Ser5^P signals were normalized with DAPI signals, and the average normalized RNA Pol II Ser5^P 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 and **p < 0.01 by Student’s t test. NS means not significant. Black arcs show comparisons between no treatment and alpha-amanitin treatment at the same cell stage. The data for GFP::LacI-tethering strain without alpha-amanitin treatment are the same as in Fig. 4. b Immunofluorescence of CENP-A^HCP−3 on first-generation ACs at different cell stages, and ACs that have been propagated for generations in GFP::LacI-tethering strain without and with alpha-amanitin treatment. Embryos were stained with antibody against CENP-A^HCP−3 (red), antibody against LacI (green) and DAPI (blue), shown separately and merged. Scale bar represents 1 μm. Quantification of IF signals. CENP-A^HCP−3 signals were normalized with DAPI signals, and the average normalized CENP-A^HCP−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 no treatment and alpha-amanitin treatment at the same cell stage. The data for GFP::LacI-tethering strain without alpha-amanitin treatment are the same as in Fig. 3a. c Quantification of AC segregation rates in GFP::LacI-tethering strain without and with alpha-amanitin treatment embryos. AC segregation rates are scored as the % of cells with segregating ACs among all dividing cells containing ACs. The number of cells (n) analyzed was indicated. *p < 0.05 and **p < 0.01 by Chi-squared test. Black arcs show comparison between without and with alpha-amanitin treatment at the same cell stage