Fig 2: Septin suppresses actin tail formation. (A) Immunoblot analysis confirms that SEPT7 siRNA treatment of HeLa cells for 72 h reduces the levels of SEPT7, SEPT2, SEPT9, and SEPT11. (B) Representative immunofluorescence images showing that loss of SEPT7 in uninfected HeLa cells has no obvious impact on the actin cytoskeleton (green). Bar, 50 µm. (C) Immunoblot analysis confirming that infection of HeLa cells with WR vaccinia virus does not alter the level of SEPT2, SEPT7, SEPT9, or SEPT11. (D) Representative images of actin tails (green) induced by WR (red labeled with B5 antibody) in HeLa cells treated with Allstar (control) or SEPT7 siRNA. Bar, 2 µm. The graphs show the quantification of the number of CEVs inducing actin tails and their length. Error bars represent SEM from three independent experiments in which a total of 30 cells were analyzed for actin tail number and 370 tails were measured for length. (E) Quantification of the velocity, directionality, time to form, and duration of actin tails in the absence of SEPT7. Error bars represent SEM from three independent experiments in which a total of >150 actin tails were measured for duration, >50 events for tail initiation from four independent experiments, and >300 actin tails for speed and directionality. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Fig 3: SEPT2 and SEPT7 regulate glycolysis in human epithelial cells. (a) Scheme of the primary metabolic routes in human epithelial cells. Arrows indicate the flux of each metabolic pathway. H6P, hexose-6-phosphate. (b,c) HeLa cells were treated with control, SEPT2, or SEPT7 siRNA sequences for 72 h. Cell lysates were prepared for LC–MS AMRT analysis. (b) Individual quantifications for indicated glycolytic metabolites, (c) individual quantifications for indicated TCA cycle metabolites. Graphs represent mean ± SEM of normalized ion counts from three independent experiments done in duplicate. *p < .05, **p < .001 as analyzed by one-way ANOVA [Color figure can be viewed at wileyonlinelibrary.com]

Fig 4: Loss of septin promotes virus release and spread. (A) Schematic depicting key events at the plasma membrane during vaccinia virus egress. A36, the actin tail nucleator phosphorylated by Src and Abl family kinases, and B5, exposed on the surface of CEV, are integral viral membrane proteins. (B) Immunoblot analysis confirms that SEPT7 siRNA treatment of A549 cells for 72 h leads to loss of SEPT7 as well as reduction in the levels of SEPT2, SEPT9, and SEPT11. (C) Images of plaques formed on A549 cell monolayers treated with control (Allstar) or SEPT7 siRNA for 72 h under liquid overlay after 3 d of infection with WR or the YdF strain of virus, which is deficient in actin tail formation. Plaque comets are seen as a diffuse spray emanating from WR plaques in liquid overlay conditions. The graphs show the quantification of plaque size (n > 190) with error bars representing the SEM from three independent experiments. (D) Quantification of virus release and total intracellular virus in A549 cells in the presence or absence of SEPT7 at 18 h after infection with WR or the YdF virus. Error bars represent SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Bar, 1 cm.

Fig 5: SEPT2, SEPT7 and p62 promote the proliferation of intracellular Shigella. (a) HeLa cells were treated with control, SEPT2, or SEPT7 siRNA sequences for 72 h. Whole-cell lysates were immunoblotted for SEPT2 and SEPT7 to show the efficiency of depletion. Actin was used as a loading control. (b) siRNA-treated cells were infected with S. flexneri str. M90T for 1 h 40 min or 4 h 40 min, then lysed and plated on LB-agar plates. Graphs represent mean fold replication ratio 4 h 40 min/1 h 40 min ± SEM from four independent experiments. (c) HeLa cells were treated with control or p62 siRNA sequences for 72 h. Whole-cell lysates were immunoblotted for p62 to show the efficiency of depletion. Actin was used as a loading control. (d) siRNA-treated cells were infected with S. flexneri str. M90T as in (b) [Color figure can be viewed at wileyonlinelibrary.com]