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

Figure 3

GBP1 is required for autophagosome formation in a localization-dependent manner. (a) GBP1-KO cells were generated using the CRISPR/Cas9 gene editing system. HeLa WT and GBP1-KO cells were treated with 20 ng/mL IFN-γ for 24 h and then collected to confirm GBP1 expression by immunoblotting. (b) Autophagosome formation in HeLa WT and GBP1 KO cells. Cells were transfected with mCherry-LC3 as an autophagosome marker and then infected with GAS for 4 h. An overview of the merging of all channels (top) and a magnified view of each channel and that of the merging of all channels (bottom) are shown. All images are representative of three independent experiments. Scale bar: 10 μm. (c) Autophagosome formation rate in GAS-infected cells. 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. (d) Percentage of autophagosome formation in GBP1-KO HeLa cells 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 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. (e) Viability of GAS in A549 WT, GBP1-KO #1, and ATG5-KO cells. In each experiment, triplicate samples were used and colonies were counted manually. Data are presented as the from four independent experiments. Asterisks indicate statistically significant differences () as determined by two-tailed Student’s -test. The same data of GBP1 KO #2 are shown in Supplementary Figure 2D.

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