SAT III knockdown reduces levels of newly incorporated CENP-A and CENP-C at centromeres. (A) Cells stably expressing SNAP-tagged CENP-A were transfected with either SAT III–LNA or scrambled (Scrl) LNA probes as a control. After transfection, SNAP signals of preexisting proteins were irreversibly blocked and rendered undetectable for further analysis. 48 h after transfection, newly synthesized SNAP-tagged proteins were stained with TMR and imaged. Images show cells 48 h after transfection. Cells were cotransfected with fluorescein-labeled dextran to visualize transfected cells. Bars, 5 µm. (B) Quantification of the intensity of centromeric SNAP-CENP-A signal as shown in A. Centromeric intensity of SNAP–CENP-A was significantly lower (P < 0.0001) upon SAT III knockdown. Depicted are the mean values from a representative experiment (n = 200 for scrambled LNA, n = 184 for SAT III–LNA). Only dextran-positive cells were analyzed. The p-value was determined using the Student’s t test. (C) Analysis of cells carrying SNAP–CENP-C construct as in A. Cells were transfected with scrambled (Scrl) LNA as a control or with SAT III–LNA. Images show cells 48 h after transfection. Cells were cotransfected with fluorescein-labeled dextran to visualize transfected cells. Bars, 5 µm. (D) Quantification of the intensity of centromeric SNAP–CENP-C signal as shown in C. Centromeric intensity of SNAP–CENP-C was significantly lower (P < 0.0001) upon SAT III knockdown. Depicted are the mean values from a representative experiment (n = 183 for scrambled LNA, n = 195 for SAT III–LNA). Data are mean ± SEM (error bars). The asterisks represent the p-value summary.