Artificial induction of autophagy restricts M. marinum proliferation.

A. GFP-Atg8-expressing cells were infected for 5 h with mCherry-expressing M. marinum wt and treated or not with AR-12 at 2.5 μM for 2 additional hours. Representative maximum projections of live imaging are shown. White arrowheads point to GFP-Atg8 recruitment to MCV. Scale bars, 10 μm; B. Quantification of the percentage of infected cells with GFP-Atg8+ bacteria at 7 hpi. Means and standard deviations from six (mock) and three (AR-12) independent replicates are represented and an unpaired t test showed no statistical significance (ns, p > 0.05). 688 and 297 infected cells were counted for the mock and the AR-12 treatments, respectively; C. Classification of types of GFP-recruitment for infected mock (258 cells) and AR-12 (141 cells) treated. Means and standard deviations from six (mock) and three (AR-12) independent replicates are represented. Unpaired t test (**p ≤ 0.01; ***p ≤ 0.001); D. Cells infected with lux-expressing M. marinum wt bacteria were treated or not with AR-12 at 2.5 μM. Intracellular bacteria growth [relative luminescence units (RLU)] is represented as the mean and standard deviation from triplicates. Statistical differences were calculated with a Bonferroni post hoc test after two-way ANOVA (*p ≤ 0.05).