Suppression of myosin VI expression leads to a hyper-proliferation of Salmonella and an accumulation of ubiquitylated Salmonella within LC3-positive autophagosomes.

<p>(<b>A</b>) HeLa cells following myosin VI siRNA or TAX1BP1, NDP52, OPTN (TNO) siRNA transfection were subjected to Salmonella infection for the indicated time points. Gentamicin protection assays were performed and colonies were counted at each time point. Results are represented as the fold replication from 2 hours, which represents the amount of bacterial proliferation. Results are from >3 independent experiments and error bars represent s.d. Western blot analysis was performed on whole cell lysates and immunoblotting was performed to the indicated proteins. (<b>B</b>) HeLa cells mock, myosin VI siRNA, or TNO siRNA treated were infected with mCherry expressing Salmonella for 8 hours, followed by processing for confocal microscopy. Cells were immunostained for ubiquitin (green). Nuclei were labelled with Hoechst (blue). The % of infected cells with ubiquitin (+) Salmonella was quantified. The results depict >3 independent experiments and the error bars represent the s.d. (<b>C</b>) HeLa cells transfected with myosin VI or TNO siRNA were infected with BFP-expressing Salmonella for 8 hours. Cells were processed for confocal microscopy and immunostained for LC3 (green) and ubiquitin (red). Scale bar, 20 μm. Quantitation at 4 and 8 hours post-infection, with BFP-expressing Salmonella, of the % of ubiquitin (+) Salmonella which are LC3 (+). Results represent 3 independent experiments and the error bars indicate the s.d.</p