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Figure 6


Fig. 6. RNAi-mediated silencing of KIFC5A causes centrosome amplification and aberrant multi-polar spindles in NIH 3T3 cells. (A1) Efficiency of silencing by quantitative real-time PCR. This shows an average knockdown of KIFC5A mRNA to 27.0±10.5% (s.d.) of control-silenced levels (silencing with medium GC content control silencing oligo; see Materials and Methods), whereas mRNA of the (unrelated) mitotic motor protein Eg5 shows 109.9±24.7% of control on average. mRNA levels of the PBGD housekeeping gene were used for sample normalisation. (A2) Efficiency of silencing by western blot. This shows a visible reduction of KIFC5A protein levels in silenced cells (lane 2; approx. 35%), compared with control-silenced cells (lane 1). Dynein detection (upper band) served as an internal loading control. (A3) Verification of silencing by triple immunofluorescence. Cells were immunolabelled for {alpha}-tubulin, KIFC5A and counterstained for DNA (red, green and blue in the overlay, respectively). Whereas the lower panels (control-silenced cell) show normal KIFC5A labelling of the spindle in prometaphase, labelling is hardly detectable in the KIFC5A-silenced cell (upper panels) at the same exposure (300 milliseconds). Bars, 5 µm. (B1) Quantification of the silencing effect on centrosome numbers in mitotic cells. KIFC5A-silenced mitotic cells contain a higher number of centrosomes per cell, 120 hours after initial siRNA treatment, compared with the control. Shown are the means from three independent experiments (see also Table 2). Bars represent s.d. values. (B2) Immunofluorescence of silenced cells, displaying an increase in the number of centrosomes and spindle poles. The top panel shows an example of a KIFC5A-silenced cell, immunostained for {gamma}-tubulin (green), {alpha}-tubulin (red) and counterstained for DNA (blue). In the bottom panel, anti-centrin, instead of {alpha}-tubulin, was used. The exact match between {gamma}-tubulin (green) and centrin (red) labelling can be observed. The extracted detail in insets (bottom) illustrates that supernumerary centrosomes consist of duplicated centrioles (arrows). Bars, 10 µm.