<i>M</i>. <i>marinum</i> induces an early autophagic response in <i>D</i>. <i>discoideum</i>.

<p><b>A.</b> Two representative maximum projections showing GFP-Atg8-expressing cells infected (bottom) or mock-infected (top) with mCherry-expressing wt <i>M</i>. <i>marinum</i>. Images were recorded live 1.5 h after infection. White arrowheads point to GFP-Atg8 structures. Scale bars, 10 μm; <b>B.</b> The median and interquartile ranges of the number of GFP-Atg8 structures per cell were calculated. For each condition, 300 cells from independent triplicates were counted. Mann-Whitney test (****<i>p</i> ≤ 0.0001); <b>C.</b> Single sections of live GFP-Atg8-expressing amoebae 1.5 h after infection with mCherry-expressing <i>M</i>. <i>marinum</i> wt. White, magenta and yellow arrowheads point to GFP-Atg8 dots, patches and GFP-Atg8-positive (GFP-Atg8⁺) MCV, respectively. Scale bars, 5 μm; <b>D.</b> At 1.5 hpi, 256 cells (100%) with GFP-Atg8⁺ <i>M</i>. <i>marinum</i> were classified as in <b>C</b>. Means and standard deviations from three independent infections are represented; <b>E.</b> EM of the different types of autophagosomes in the vicinity of the MCV at 7 hpi. White asterisks label bacteria, white arrowheads point to round phagophores and autophagosomes, magenta arrowheads point to extended autophagosomes, and the yellow arrowhead indicates a double membrane compartment containing mycobacteria. Nuclei . Scale bars, 1 μm; <b>F.</b> Sections (top) and maximum projections (bottom) showing co-localization (white arrowheads) of autophagy markers with bacteria at 1–1.5 hpi. Around 50% and 30% of the Atg8-positive bacteria were also positive for Ub and GFP-p62, respectively. Scale bars, 5 μm; <b>G.</b> qPCR results of relative abundance of <i>atg8a</i>, <i>atg8b</i>, <i>atg1</i> and <i>p62</i> mRNAs at 3 hpi. The mRNA level in non-infected cells is indicated as 1. The mRNA level of the housekeeping gene <i>gapdh</i> was used for normalization. Means and standard deviations from three independent experiments performed in triplicates are represented.