The impact of septin depletion on HeLa cells. A, Western
blot (WB) of HeLa cells transfected with siRNA against control
(CTRL), SEPT9, or SEPT11. Cell lysates were separated by 10% SDS-PAGE
before immunoblotting. The blots were probed with antibodies specific to
GAPDH, SEPT9, SEPT11, and actin. GAPDH is shown as a loading control. The
red box outlines depleted protein levels for targeted septins.
B, septin depletions have different effects on cell shape and actin
and septin filament distribution. Endogenous F-actin and SEPT9 were visualized
by immunostaining with anti-F-actin (green) and anti-SEPT9 antibodies
(red) for control (CTRL)-, SEPT9-, and SEPT11-depleted
cells. Nuclei were marked with DAPI (blue). Scale bars
indicate 10 μm. C, different classes of SEPT9 filament
distribution. In siRNA-treated HeLa cells, septin and actin filaments were
distributed into different patterns. Cells presenting filaments around the
cell periphery and under the nucleus were designated as Class 1, cells with
filaments only around the cell periphery, but not under the nucleus were
designated as Class 2, and cells no longer presenting any filaments were
designated as Class 3. D, different effects of septin inactivation on
the organization of SEPT9 filaments. Confocal Z-stack images of siRNA-treated
cells were categorized by a filament tracking algorithm into the three
pre-assigned classes of filament distribution (Fig. 1C). The
percentage of septin-depleted cells observed to still have filaments,
i.e. % of cells in Class 1 and Class 2 versus % of cells in
Class 3, was statistically compared with expectations as derived from control
cells by chi square test (p value(C1+C2 versus
C3)). To assess whether SEPT11-depleted cells retain SEPT9 filaments
under the nucleus, the distribution of SEPT9 filaments in SEPT11-depleted
cells, i.e. % of cells in Class 1 versus % of cells in Class
2, was statistically compared with expectations as derived from control cells
by chi square test (p value(C1 versus C2)).