Septins are required for apoptosis induced by Shigella infection. (a) HeLa cells were transfected with control or SEPT7 siRNA for 72 hr. Cells were then infected with S. flexneri M90T or the S. flexneri ΔgalU mutant for 4 hr. Whole-cell lysates were immunoblotted against caspase-3, caspase-7, and caspase-9. GAPDH was used as a loading control. Data are representative of three independent experiments. (b) Schematic representation of CRISPR/Cas9 targeting of sept2. Three crisprRNA (crRNA, green arrows) were designed, targeting three consecutive exons (red boxes) of the zebrafish sept2 gene. (c) Representative images of control (CTRL) and sept2 crispants. Lines indicate a difference in overall body length between CTRL and sept2 crispants. Images were acquired using Leica M205 FA stereomicroscope. (d) Quantification of difference in body length between CTRL and sept2 crispants. Bars represent the mean ± SEM from three independent experiments (n = 12 larvae per bar). **p < .01 by Student's t test. (e) Survival assays of CTRL and sept2 crispants injected with approximately 9,000 CFU of either S. flexneri WT or S. flexneri ΔgalU. **p < .01, ****p < .0001 by log–rank (Mantel–Cox) test. Curves are cumulative of 3 independent experiments (n = 53–85 larvae per curve). Statistical tests for survival curves were performed using the log–rank (Mantel–Cox) test (which intrinsically take into consideration the overall trend of survival over the different times of observation), and are indicated for overall survival. (f) CFU enumeration of CTRL and sept2 crispants injected with approximately 9,000 CFU of either S. flexneri WT or S. flexneri ΔgalU. CFU were collected at 0, 6, and 24 hr post-infection, and are indicated on the X-axis. Bars represent the mean ± SEM from three independent experiments (n = 15 larvae per bar). *p < .05, ****p < .0001 by one-way ANOVA. Statistical tests for CFU counts were performed using one-way ANOVA, and are indicated for the individual timepoints
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