Extended Data Fig. 2: Influence of HD5 on cytoskeleton of different cell lines and HeLa mutants.
a, Fluorescence microscopy analysis and quantification (n = 10) of cytoskeleton alterations in 10 adherent epithelial cell lines, primary colonocytes and 8 suspension cell lines in the absence (control) or presence of 2 μM HD5. In the upper panel, for epithelial cell lines and primary cells, the bar represents 10 μm; for suspension cells, the bar represents 20 μm. HIFE are indicated with arrows. F-actin is grey and nuclei are blue (DAPI). In the lower panel, bars indicate mean; errors, s.d. b, Fluorescence microscopy analysis of cytoskeleton of HeLa cells microinjected with HD5. HD5 was microinjected into HeLa cells to produce intracellular concentrations of 0.5 to 5 μM, which failed to produce any significant alteration to the cytoskeleton. Injected cells are indicated in red (with Texas Red as microinjection tracker), F-actin is green and nuclei are blue (DAPI). The bar represents 50 μm. c, Fluorescence absorbance analysis of in vitro actin polymerization dynamics of 4 μM actin in the presence of 4 μM HD5 or 1 nM formin. The addition of up to 4 μM HD5 into an in vitro actin polymerization system had minimal effect on the nucleation and elongation of actin filaments. Each data point represents the fluorescence value at that time point. d, Immunoblotting examination of β1 integrin and CD44 knockout in HeLa cells. e, Influence of β1 integrin and CD44 knockout on Shigella adhesion/invasion (n = 6). Data are presented as mean±s.d. f, Influence of β1 integrin and CD44 knockout on the cytoskeleton of HeLa cells in the presence or absence of 4 μM HD5. The bar represents 20 μm. Depletion of β1 integrin subunit and CD44 from HeLa cells by the CRISPR/Cas9 system neither affected the association of HD5 with Shigella adhesion and invasion, nor the formation of filopodia. Significance was determined by a two-tailed unpaired Student’ s t-test, and indicated as the p value. ns: no signification, **p < 0.01, ***p < 0.001 and ****p < 0.0001. All results are representative of at least three independent experiments.
