Fig. 4
GABAergic autophagy activation promotes phagosomal maturation against mycobacterial infection. a–d BMDMs were infected with Mtb-ERFP (MOI of 5) for 4 h, incubated with GABA (100 μM), muscimol (Mus; 100 μM), or isoguvacine hydrochloride (Iso; 100 μM) for 18 h, then stained with LC3 (green; a, b) or LAMP2 (green; c, d), and DAPI (for nuclei; blue). a Cells were visualized by confocal microscopy. Scale bars, 5 μm. b Quantitative data of colocalization of Mtb-ERFP and LC3 per cell. c Cells were visualized by confocal microscopy. Scale bars, 5 μm. d Quantitative data of colocalization of Mtb-ERFP and LAMP2 per cell. e–h Mice were injected i.v. with Mtb (1 × 106 CFU), treated with PBS or GABA (daily i.p. 200 mg/kg), and monitored at 14 days post-infection (dpi). e Representative low- (top) and high-magnification (bottom) transmission electron micrographs of lung tissues. Scale bars, 1 μm (top) and 200 nm (bottom). f Quantitative data of phagosomes and autophagosomes containing Mtb. g Representative immunoblots for the expression of the indicated proteins from lung tissues. h The densitometric values for p62 were normalized to β-tubulin. i Atg7f/fLysM-Cre- (Atg7 WT) and Atg7f/fLysM-Cre+ (Atg7 KO) BMDMs were infected with Mtb (MOI of 5) and then treated with GABA (100 μM). After 3 days, intracellular survival of Mtb was assessed by CFU assay. *p < 0.05, and ***p < 0.001. ns: not significant, UI: uninfected, SC: solvent control (0.1% PBS). Statistical significance was determined by one-way ANOVA (b, d, h), Mann–Whitney U-test (f), or two-way ANOVA (i). Data shown (means ± SEM) represent the combined results of triplicates from three independent experiments (b, d, f, h, i). Images are representative of three (e, g) or four (a, c) independent experiments
