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. 2020 Mar 9;16(3):e1008364. doi: 10.1371/journal.ppat.1008364

Fig 3. GBPs inhibit Arp2/3-dependent actin motility required for MNGC formation.

Fig 3

Unprimed BMDMs were infected (MOI 5) with B. thailandensis (GFP+) or the fusion-defective mutant as described and after the final wash, CK-666 (200 μM) was added where indicated. Phase-contrast images were collected at 20 h post-infection with (a) wildtype B. thailandensis or (b) B. thailandensis vgrG5 ΔCTD, with or without CK-666. Dotted yellow lines indicate multinucleated cells. (c) Uptake of Sytox Green at 20 h post-infection in CK-666 treated BMDMs. Fluorescent microscopy images of BMDMs infected with (d) wildtype B. thailandensis or (e) B. thailandensis vgrG5ΔCTD, with or without CK-666 were collected at 6 h post-infection. (f) Super-resolution microscopy images were collected from 6 h samples. (g) The percentage (%) actin tail-positive wildtype bacteria (indicated by empty yellow arrow in (d) and (e)) or B. thailandensis vgrG5ΔCTD (stained with DAPI, indicated by white arrows) were quantified from at least three fields per condition normalized to total bacterial counts. Significance was determined by one-way ANOVA with the Holm-Sidak multiple comparisons test, ****P < 0.00001, ***P < 0.0001 (c, g). Images and quantification are representative of (a,b,c) at least three independent repeats, (d,e,f) one representative of three experiments, or (g) quantified from at least three fields from one independent experiment.