Figure 4. GTP-locked Irgb10^K81A mutant but not wildtype Irgb10 targets C. trachomatis inclusions in Atg3- and Atg5-deficient cells with high efficiency.

(A) Atg5^−/− MEFs were transfected with GFP-fusion constructs expressing either wildtype Irgb10 (WT) or the Irgb10^S82N mutant that is deficient of GTP binding. Cells were subsequently infected with C. trachomatis and treated with 200 U/ml of IFNγ at 3 hpi. Fixed cells were stained with Hoechst. Representative images are shown. (B) WT, Atg3^−/− & Atg5^−/− MEFs were transfected with the indicated constructs. Cells were infected with C. trachomatis and treated with IFNγ at 3 hpi. Cells were fixed at 20 hpi and stained for the C. trachomatis inclusion membrane marker IncG as well as DNA (Hoechst). Representative images are shown. (C) Graphical representation of the frequency at which WT Irgb10 and the Irgb10^K81A mutant colocalize with inclusions. Average values ± SD of three independent experiments are shown. Differences in the targeting frequency for WT Irgb10 and Irgb10^K81A to inclusions were evaluated for statistical significance (**, p<0.005).