Guanylate-Binding Protein 1 Regulates Infection-Induced Autophagy through TBK1 Phosphorylation

Figure 4

GBP1 is involved in p62 recruitment to GAS. (a) Recruitment of autophagy-related proteins to invading GAS. Cells were transfected with the indicated plasmid prior to infection or labeled with antibodies for the indicated protein after infection. Cellular and bacterial DNA was stained with DAPI. All images are representative of three independent experiments for 2 h of infection. Insets: enlargements of indicated areas. Scale bar: 10 μm. (b) Percentage of indicated protein recruitment to GAS in (a). Cells were infected with GAS for 2 or 4 h. Data are presented as the from three independent experiments (100 infected cells were manually counted in each experiment). Asterisks indicate statistically significant differences () as determined by two-tailed Student’s -test. (c) Percentage of p62 recruitment to GAS under conditions of GBP1 mutant overexpression. HeLa WT and GBP1-KO cells were transfected with EmGFP, EmGFP- GBP1 intact, or mutants. Data are presented as mean from three independent experiments (100 infected cells were manually counted in each experiment). Asterisks indicate statistically significant differences () as determined by two-tailed Student’s -test. (d) Degradation of the lysosome damaged by LLOMe treatment. HeLa cells were treated with 0.5 mM LLOMe for 2 h. Damaged lysosomes were labeled with an anti-galectin-3 antibody. Scale bar: 10 μm. (e) Starvation-induced autophagosome formation in HeLa WT and GBP1-KO cells. Indicated cells stably expressing mCherry-LC3 as an autophagosome marker were incubated in the medium without serum for 2 h. All images are representative of three independent experiments. Scale bar: 10 μm. (f) Quantification of mCherry-LC3 puncta in (e). At least 30 cells were obtained from five randomly selected fields in each condition (repeated three times). Puncta were counted automatically using the Analyze Particle plugin in Fiji software (ImageJ; National Institutes of Health).

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