Extended Data Fig. 1: Influence of HD5 and Shigella on cytoskeleton in HeLa cells and schematic illustration of experimental procedure of Shigella infection and FRET assay.
a, Fluorescence microscopy analysis of cytoskeleton of HeLa cells in the presence of a serial concentration of HD5. F-actin is red, and nuclei are blue (DAPI). The bars represent 20 μm. b, Influence of Shigella at MOI of 100 on the cytoskeleton of HeLa cells. F-actin is red, GFP-expressing Sf301 is green and nuclei are blue (DAPI). The bars represent 10 μm. c, Schematic illustration of experimental procedure of different pretreatment combinations and co-incubation. To further distinguish the effects of HD5 on bacteria and host cell respectively, HD5 was allowed to interact with Shigella or HeLa cells separately for 15 min, followed by 3 washes to thoroughly remove unbound defensin molecules. Different pretreatment combinations result in different levels of bacterial adhesion and invasion as shown in Fig. 2c. d, Illustration of Förster resonance energy transfer (FRET) assay and its application to analyze the T3SS activity of Shigella in its invasion in the absence of presence of HD5. e, Influence of 2 μM HD5 on the adhesion and invasion of Shigella ΔmxiD mutant in comparison with wildtype Sf301 strain. Significance compared with solvent control (0 μM) group was calculated using a one-way ANOVA (Tukey’s multiple comparison Test), and indicated as the p value. ***p < 0.001 and ****p < 0.0001. Data are presented as mean±s.d. Results are representative of at least three independent experiments.
