Fig. 2
Four-lysine mutations in HJURP R1 or R2 disturb HJURP localization and CENP-A deposition. a Schematic of the CENP-A deposition experiment for testing functionality of HJURP variants. SNAP-Cell 647-SiR was used to label newly produced SNAP-CENP-A at the time point +4 h. b, c Representative images showing SNAP-CENP-A fluorescence and EGFP-NLS or EGFP-HJURP variants in fixed HeLa cells treated as described in panel a. Centromeres were visualized with CREST sera. Control cells were treated with transfection reagent (Lipofectamine RNAiMAX) in the absence of HJURP siRNA. DIC, differential interference contrast. One side of the white square in the DIC panel represents 20 µm. White scale bars indicate 10 µm. All cell biological experiments in this paper were repeated at least three times. d Quantification of the centromere fluorescence intensity of SNAP-CENP-A. Centromere spots were detected using the images of CREST channel and were applied to the images of other data channels. In each experiment, a mean value of centromere fluorescence intensity was obtained from at least 340 centromere spots from at least 20 early G1 cells. The highest 10% and the lowest 10% intensity values were considered outliers and excluded. The bar graph represents mean values from the three replicate experiments (blue dots indicate the mean values from each experiment). Error bars indicate standard deviations. The ways of quantification and representation described here apply to other figures displaying experiments in HeLa cells. e Quantification of the centromere fluorescence intensity of EGFP signal. Source data are provided as a Source Data file
