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. 2015 Jan 1;128(1):171–184. doi: 10.1242/jcs.163659

Fig. 2.

Fig. 2.

CENP-Q is required to load CENP-E to kinetochores. (A) Immunofluorescence microscopy images of a metaphase HeLa E1 cells treated with control siRNA, or siRNA against CENP-E (CENP-E siRNA) or CENP-Q for 48 h and then stained with CREST antisera (red) and antibodies against CENP-E (green) and α-tubulin (blue). The image is a z-projection (10 focal planes at 0.2 µm spacing). Yellow arrows point to unaligned kinetochores. Insets show single kinetochore pairs. (B) Immunofluorescence microscopy images of metaphase HeLa E1 cells treated with control, CENP-E or CENP-Q siRNA for 48 h and stained with CREST antisera (red) and antibodies against CENP-Q (green) and α-tubulin (blue). The image is a z-projection (10 focal planes at 0.2 µm spacing). Yellow arrows point to unaligned kinetochores. Insets show single kinetochore pairs. (C) Left panel, quantification of CENP-E immunofluorescence levels in HeLa E1 cells treated with control siRNA, CENP-E siRNA or CENP-Q siRNA for 48 h. The intensities are determined at each kinetochore relative to that of CREST after background subtraction, n≥150 kinetochores per condition from three independent experiments. Dashed line indicates CENP-E levels in control siRNA cells. Error bars indicate ±s.d. Right panel, quantification of CENP-Q immunofluorescence levels in HeLa E1 cells treated with control siRNA, CENP-E siRNA or CENP-Q siRNA for 48 h. Intensities are determined at each kinetochore relative to that of CREST after background subtraction, n≥100 kinetochores per condition from two independent experiments. Dashed line indicates CENP-Q levels in control cells. Error bars indicate ±s.d. (D) Immunofluorescence microscopy images (z-projections, 10 focal planes at 0.2 µm spacing) of a CENP-Q siRNA rescue experiment in HeLa E1 cells. Cells were treated with CENP-Q siRNA or control siRNA for 12 h and then transfected with an siRNA-resistant plasmid expressing CENP-Q–eGFP or a control eGFP expression plasmid for a further 48 h. To reduce the effect of the mitotic stage on alignment, cells were arrested in metaphase with MG132 at a 1 µM final concentration for 90 min, followed by fixation. Cells were stained with antibodies against CENP-A (blue) and CENP-E (red). (E) Quantification of CENP-E immunofluorescence levels in the CENP-Q siRNA rescue experiment detailed in D. Intensities were determined at each kinetochore relative to that of CENP-A after background subtraction, n≥150 kinetochores per condition from three independent experiments. Dashed line indicates CENP-Q levels in cells treated with the control siRNA. Error bars indicate ±s.d. Scale bars: 3 µm (A,B,D).