Fig. 6
GABARAPL1 is essential for phagosomal maturation and antimicrobial responses in macrophages. a BMDMs infected with Mtb (4 h) were incubated with GABA (100 μM), muscimol (Mus; 100 μM), or isoguvacine hydrochloride (Iso; 100 μM) for 6 h, followed by qRT-PCR analysis for Gabarapl1 and Gabarapl2. b, c RAW264.7 cells were transfected with mock or EGFP-Gabarapl1 plasmid for 24 h and infected with Mtb-ERFP. Cells were fixed and stained with LAMP2 (cyan). b Cells were visualized by confocal microscopy. Confocal z-stack images were obtained and reconstituted to three dimensions. Scale bars: 10 μm (top), 5 μm (middle), and 2.5 μm (bottom). b' shows different angles of the models. c Quantitative data of colocalization of Mtb-ERFP, LAMP2, and GABARAPL1 per cell. d RAW264.7 cells were transfected with mock or Gabarapl1 plasmid for 24 h and infected with Mtb for the indicated times. Intracellular survival of Mtb assessed by CFU assay. e–g BMDMs were transduced with lentivirus expressing shNs or shGabarapl1 using polybrene (8 μg/ml). e, f After 36 h, BMDMs were infected with Mtb-ERFP for 4 h and incubated with GABA (100 μM) for 18 h. e Semiquantitative PCR analysis was performed to assess transduction efficiency (top). Cells were visualized by confocal microscopy (bottom). Scale bars, 5 μm. f Quantitative data of colocalization of Mtb-ERFP and LAMP2 per cell. g Intracellular survival of Mtb assessed in BMDMs treated with GABA (10, 100, and 1000 μM) for 3 days. h BMDMs from WT or Gabarapl1 KO were infected with Mtb and treated with GABA (100 μM) or Mus (100 μM) for 3 days. Intracellular survival of Mtb assessed by CFU assay. *p < 0.05 and **p < 0.01. U: untreated, SC: solvent control (0.1% PBS), ns: not significant. Statistical significance was determined by one-way ANOVA (a, g, h), Mann–Whitney U-test (c), and two-way ANOVA (d, f). Data shown (means ± SEM) represent the combined results of duplicates (a, h) or triplicates (c, d, f, g) from three independent experiments. Images are representative of three independent experiments (b, e)
