Fig. 6.

VP16, but not p65 tethering, negatively affects CENP-A loading at the HAC centromere. (A) Schematic diagram of quench–pulse-chase experiments in cells co-transfected with SNAP-tagged CENP-A and either of the tetR fusion constructs to determine the centromeric loading of newly synthesized CENP-A. (B–D) Cells expressing the indicated tetR fusion construct (green, panel 1) and pulse-labelled with TMR-Star (red, panel 2). Merged images (panel 3) represent the overlay of EYFP signal with TMR and DAPI staining (blue). Arrowheads depict the HAC. Scale bar: 5 μm. (E) Quantification of HAC-associated TMR-Star signal in cells as in B–D. AFU values are plotted relative to the average TMR-Star signal measured at endogenous centromeres. Solid lines indicate the median. ns, no significant difference.