Stress Granule Formation During Shigella Infection of Cancerous Epithelial Cells

Begin with a multi-well plate containing adherent cancerous epithelial cells on a coverslip.

Add Shigella flexneri, a pathogenic bacterium.

Centrifuge the plate to promote bacterial contact with the cells, then incubate.

Using a specialized secretion system, Shigella injects virulence proteins into the epithelial cells.

These virulence proteins modulate the epithelial signaling system, facilitating bacterial uptake.

Inside the cells, the bacteria continue secreting virulence proteins, which disrupt cellular translation and accumulate untranslated mRNAs.

RNA-binding and signaling proteins aggregate with the untranslated mRNAs, forming membrane-less cytoplasmic granules known as stress granules.

Discard the media and wash to remove unattached bacteria.

Next, add pre-warmed culture media supplemented with gentamicin to eliminate surface-attached bacteria.

Add a fixative to preserve cellular structures and stress granules.

Discard the fixative and wash with gentle shaking to remove residual fixative.

The cells are ready for immunofluorescence staining to visualize Shigella-induced stress granules.

Replace the wash with 500 microliters of infection medium per well, and centrifuge the 24-well plate