LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute
LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute
LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute
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analysis. (C) The mean fluorescence intensities of the cell were measured for 24 cells in 3<br />
independent experiments for each immunofluorescence analysis. (Original magnification<br />
x 630 with 2X optical zoom).<br />
4.3.14 Generation of HCT116 derived KIF5B knockdown cell lines.<br />
The experiments done so far suggested that the recruitment of PG to cell border is<br />
microtubule dependent. We next wanted to understand whether KIF5B is required for the<br />
recruitment of PG and other desmosomal proteins to the cell border. To test the<br />
hypothesis that PG recruitment to the cell surface thus initiating desmosome formation is<br />
dependent on Kinesin 1 motor protein (KIF5B), the expression of KIF5B was inhibited in<br />
HCT116 cells using vector driven RNA interference. HCT116 cells were transfected with<br />
the KIF5B knockdown construct or the vector control and stable clones were generated<br />
after selection in puromycin. As compared to the control pLKO.1 vector we identified<br />
five stably selected clones with a down regulation of KIF5B (Figure 4.3.25 (A)) as<br />
determined by Western blotting using specific antibodies to KIF5B. A Western blot for<br />
actin served as a loading control. It is reported previously that when the Kif5b gene was<br />
disrupted in mice, perinuclear accumulation of mitochondria is observed, suggesting that<br />
the KIF5 motors are essential for mitochondrial transport (350). To confirm whether<br />
these mitochondrial defects are replicated in our HCT116 derived KIF5B knockdown<br />
clones, we incubated the vector control cells and two KIF5B knockdown clones K1.3 and<br />
K1.5 with Mitotracker488 (500nM), which localizes to mitochondria, for 45 minutes<br />
followed by confocal imaging in live cells. As shown in the figure 4.3.25 (B), both<br />
HCT116 derived KIF5B knockdown clones K1.3 and K1.5 show a perinuclear<br />
accumulation of mitochondria, In contrast, the mitochondria in vector control cells are<br />
present all through the cell. This result suggests that the KIF5B knockdown clones<br />
generated by us functionally affects the transport of mitochondria.<br />
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