Fig. 3
GABAergic activation triggers autophagy in macrophages. a Heatmap analysis of the RNAseq data (representative of duplicate determinations). BMDMs were treated with GABA (100 μM) for the indicated times and then subjected to RNAseq analysis. b BMDMs were treated with GABA (100 μM) for 6 h. Cells were subjected to qRT-PCR to determine mRNA levels of autophagy genes. c BMDMs were treated with GABA (10, 50, or 100 μM) for 18 h and the expression of LC3B was analyzed by flow cytometry (horizontal axis, fluorescence (FITC) intensity). Representative gating strategy is shown in Supplementary Fig. 5. d, e BMDMs were incubated with GABA (100 μM), muscimol (Mus; 100 μM), or isoguvacine hydrochloride (Iso; 100 μM) and then stained with LC3 (green) and DAPI (nuclei; blue). d Confocal microscopic analysis. Scale bars, 5 μm. e Quantitative data of LC3 punctate analysis. f BMDMs were pretreated with or without bafilomycin A1 (BafA, 200 nM; 2 h) and incubated with GABA (10, 50, or 100 μM), Mus (100 μM), or Iso (100 μM) for 18 h. LC3 and β-tubulin levels were evaluated by immunoblotting. g, h BMDMs were incubated with GABA (100 μM) for 24 h. g Representative transmission electron micrograph of BMDMs. Scale bars, 1 μm (left), 500 nm (middle), and 200 nm (right). h Quantitative data of autophagic vesicle numbers. i, j BMDMs were transduced with a retrovirus expressing a tandem LC3B plasmid (mCherry-EGFP-LC3B). Cells were pretreated without or with BafA (200 nM; 2 h) and incubated with GABA (100 μM) for 18 h. i Confocal microscopic analysis for LC3. Scale bars: 5 μm. j Quantification of yellow puncta/total red puncta (%) per cell. *p < 0.05, **p < 0.01, and ***p < 0.001. U: untreated, SC: solvent control (0.1% PBS, for c–e, g, h; 0.1% dimethyl sulfoxide, for f, i, j), N: nucleus. Statistical significance was determined by Mann–Whitney U-test (b), one-way ANOVA (e, j), or unpaired t-test (h). Data shown (means ± SEM) represent the combined results of triplicates from three independent experiments (b, e, h, j). Images are representative of three or four (a, c, d, f, g, i) independent experiments
