Fig. 6. Depletion of human SPT6 leads to the loss of CENP-A maintenance.

a Experimental setup. HeLa cells expressing SNAP-tagged CENP-A were treated with TMR-star to detect previously incorporated CENP-A and siRNA-treated to deplete proteins indicated in (b, c). Cells were then synchronized in S phase by a thymidine block and released. Cells were allowed transit through G1 phase and were collected at the next G1/S boundary by re-addition of thymine. b Cells were treated with indicated siRNAs for 48 h and extracts were processed for immunoblotting and probed with indicated antibodies. CC CENP-C, M Marker. N = 3 independent experiments. c Representative images of siRNA-treated cells, 48 h after TMR pulse labeling and mRNA depletion. Cells were counter stained with DAPI and anti-CENP-B antibodies to label DNA and centromeres, respectively. Scale bar represents 10 μm. d Quantification of experiments shown in (a, c). Mean and ± SEM of N = 3 independent experiments is shown normalized to median control siRNA (ctrl). Images are quantified with CrAQ2. n_Crtl = 3989, n_CC = 4097, n_{Spt6_1} = 3635, n_{Spt6_1} = 3559 centromeres. Statistical significance: triple dots P_CC = 0.0001, single dots P₁ = 0,0374, single dots P₂ = 0.019. (One-way ANOVA, Dunnett’s multiple comparisons test). Source data are provided as a Source Data file.