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. 2017 Sep 4;214(9):2611–2628. doi: 10.1084/jem.20160999

Figure 3.

Figure 3.

Impaired xenophagy in macrophages from GRN−/− mice. (A) L. monocytogenes clearance assay of BMDMs from GRN+/+ (blue circles) and GRN−/− mice (red triangles) in response to the indicated strain of L. monocytogenes. n = 3 independent experiments, with each experiment performed in quadruplicate (mean value of each independent experiment plotted in graph). ***, P < 0.001 versus GRN+/+, using a linear least squares regression analysis. (B) Representative micrographs from n = 5 independent experiments showing BMDMs from GRN+/+ (top row) and GRN−/− (bottom) mice infected with GFP-labeled ΔactA L. monocytogenes (left) and stained for LC3 (middle). Example bacteria are marked with an arrowhead. Bars, 10 µm. (C) Quantification of L. monocytogenes/LC3 colocalization from experiments as shown in B. n = 5 independent experiments. **, P < 0.01 versus GRN+/+ using an unpaired t test. (D) Representative Western blot from n = 4 independent experiments of cell lysates from GRN+/+ and GRN−/− BMDMs starved for the indicated time in the absence (top row) and presence (middle row) of 100 nM bafilomycin A1 and probed for LC3 and actin. The actin (loading control) immunoblots shown in Figs. 3 D and S2 A are identical; this actin immunoblot, as well as LC3 (Fig. 3 D) and p62 (Fig. S2 A), were obtained by reprobing of the same Western blot. Molecular mass is indicated in kilodaltons. (E) Quantification of LC3 II levels from experiments as in D. n = 4 independent experiments. §, P = 0.0028 for GRN−/− versus GRN+/+ using two-way ANOVA and Dunnett’s multiple comparisons test (0.5 h = P < 0.01, 1 h = P < 0.05, 2 h = P < 0.0001, and 4 h = P < 0.01). (F) Representative images from n = 3 independent experiments of GRN+/+ (left) and GRN−/− (right) BMDMs treated with the indicated compounds before staining for LC3. Bars, 10 µm. (G) Quantification of number of LC3 puncta per cell from experiments as shown in F. n = 56, 47, 39, 60, 67, 65 cells from three independent experiments for the groups listed left to right. **, P < 0.01 versus GRN+/+ using a Mann–Whitney U test. Error bars represent mean ± SEM. EBSS, Earle’s balanced salt solution.