Figure 4. LSD2 activity at the alphoid^tetO HAC centromere affects kinetochore function and leads to chromosome segregation defects.

(a) Representative immunofluorescence images of mitotic 1C7 cells expressing the tetR-EYFP-LSD2^WT fusion protein and stained for CENP-A. Images show examples of normal (top) and abnormal alphoid^tetO HAC segregation (middle and bottom rows). Arrowheads depict the HAC. Scale bar, 10 μm. (b) Analysis of the frequency of normal and abnormal alphoid^tetO HAC segregation at the indicated time points. Data represent the mean (and s.e.m.) of three independent assays of each time point after doxycycline washout (n=25 mitosis per time point and experiment; 0 days versus each time point, **P<0.0001; χ²-test). (c) Quantification of alphoid^tetO HAC copy-numbers as determined by the EYFP spot in interphase nuclei. Data represent the mean (and s.e.m.) of three independent assays of each time point after doxycycline washout (n=1,000 nuclei per time point and experiment; 0 days versus each time point, **P<0.0001; χ²-test). (d) Analysis of the frequency of normal and abnormal alphoid^tetO HAC segregation in the presence and absence of selection (Blasticidin) for 10 days. The alphoid^tetO HAC was identified by in situ tetR-EYFP tethering (see Supplementary Fig. 2). Data represent the mean (and s.e.m.) of three independent assays.