Extended Data Fig. 6 |. 25HC suppresses spread of S. flexneri through mobilization of accessible cholesterol.

a, S. flexneri invades epithelial cells and disseminates from cell-to-cell. However, compared to L. monocytogenes, S. flexneri uses different molecular mechanisms and virulence factors. To test whether 25HC can inhibit S. flexneri by modulating accessible cholesterol, we carried out plaque-forming assays coupled with cholesterol repletion. HEK293A were treated with 25HC (5 μM) or vehicle for 16 hours, then 1 hour prior to S. flexneri infection, cells were treated with Chol/MCD complexes diluted in media (40 μM), or vehicle. Plaques were analysed 72 hours after avicel overlay. Representative images of three independent experiments are shown. Scale bar, 1 mm. b, Plaque area was quantified for assay described in (a), and normalized to vehicle-treated cells. Bars represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined before normalization by one-way ANOVA compared to vehicle with Dunnet’s correction.