Alphoidhybrid HAC shows epigenetically distinct centromeric
domains. (a) Representative images of HT1080–5B10 cells expressing
the indicated tetR (first panel) and lacI-fusion proteins (second
panel) and stained with antibodies recognizing H3K9me3 (third panel)
and CENP-A (fourth panel). Merged images represent the overlay of
TMR-SNAP, GFP, and H3K9me3 (MERGE 1; fifth panel) and GFP, H3K9me3
and CENP-A (MERGE 2; sixth panel). (b) Quantification of HAC-associated
CENP-A staining in individual cells transfected with the indicated
fusion proteins and plotted as A.F.U. Solid bars indicate the medians,
and error bars represent the s.e.m. n = two independent
experiments for each time point and staining. Asterisks indicate a
significant difference (**P < 0.01; Mann–Whitney
test). (c) Quantification of alphoidhybrid HAC copy-numbers
as determined by counting the GFP and/or TMR-SNAP spot in interphase
nuclei of cells transfected with the indicated fusion proteins. Data
represent the mean (and s.e.m.) of three independent assays of each
time point after doxycycline washout (n = 1,000 nuclei
per condition; *P < 0.05, **P < 0.0001; χ2-test). (d) ChIP-qPCR analysis in
HT1080–5B10 cells using the indicated antibodies. The α21-I-tetO
(tetO), α21-II-lacOgal4 (lacOgal4) hybridHAC domains,
the satellite D17Z1 (Chr17), and the degenerate satellite type-II
(Sat2) repeats were assessed. Scale bars = 10 μm.