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

Figure 2

GBP1 localization is dependent on membrane damage and its recognition by galectin-3. (a) Confocal micrographs of GBP1 localization with infection of GAS wild type (WT) or slo deletion mutant. HeLa cells overexpressing EmGFP-GBP1 were infected with the indicated bacteria for 4 h. Cellular and bacterial DNA was stained with DAPI. 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. Scale bar: 10 μm. (b) GBP1 and galectin-3 localization in GAS-infected cells. HeLa cells were transfected with EmGFP-GBP1. After GAS infection for 4 h, cells were stained with anti-galectin-3 and DAPI. An overview of the merging of all channels (top) and a magnified view of each channel and that of the merging of EmGFP-GBP1 and galectin-3 (bottom) are shown. Scale bar: 10 μm. (c) GBP1 recruitment in galectin-3-knockout cells [50]. WT and galectin-3-knockout (KO) HeLa cells were transfected with EmGFP-GBP1, followed by GAS infection for 4 h. Cellular and bacterial DNA was stained with DAPI. An overview of the merging of all channels (top) and a magnified view of each channel and that of the merging of DAPI and EmGFP-GBP1 (bottom) are shown. Scale bar: 10 μm. (d) Quantification of GBP1 recruitment in (c). Cells were manually counted by observing immunofluorescence using confocal microscopy. Data are presented as the from three independent experiments (100 infected cells were examined in each experiment). Asterisks indicate statistically significant differences () as determined by two-tailed Student’s -test. (e) Quantification of GBP1 recruitment in galectin-3 complementation. Cells were transfected with the indicated plasmid, infected with GAS for 4 h, and stained with DAPI. Cells were manually counted by observing immunofluorescence using confocal microscopy. Data are presented as the from three independent experiments (50 infected cells were examined in each experiment). Asterisks indicate statistically significant differences () as determined by two-tailed Student’s -test. (f) GBP1 recruitment to the damaged lysosome after _L-leucyl-_L-leucine methyl ester (LLOMe) treatment. HeLa cells transfected with the indicated plasmids were treated with 0.5 mM LLOMe for 2 h. Cellular and bacterial DNA was stained with DAPI. Scale bar: 10 μm.

(a)

(b)

(c)

(d)

(e)

(f)