Figure 5: Genetic requirements for ROS and ETs formation and fruiting body sterility.

(a) In situ ROS production (DHE staining, red) in wild-type (AX4, AX2) and mutant (TirA-KO, NoxABC-KO) slugs was visualized by confocal microscopy and quantified after 3D reconstruction by counting the number of red foci per slug, the P values were calculated using the student t-test, n>14. See also Supplementary Movie 6. (b) On LPS stimulation, ETs production from disaggregated slug cells from wild types and mutant strains were quantified by counting the extracellular DNA fibres stained by SYTO9 or PI, n=3. (c) K.p. c.f.u. generated from isolated fruiting bodies from wild types and mutant strains. (d) In wild-type slugs (upper), S cells not only kill engulfed bacteria, but also produce ETs to trap and kill extracellular bacteria, leading to the formation of sterile fruiting bodies. Deficiency of ET formation in mutant strains (lower) results in inefficient bacteria clearance and contaminated fruiting bodies. Scale bar, 100 μm.