Figure 4.

AMPK activation is required for OMS-induced phagosomal maturation and antimicrobial responses. (a and b) BMDMs were treated with OMS-A (10 μM; for a) or OMS-B (10 μM; for b) for indicated times (0–18 h). The cell lysates were subjected to immunoblot analysis of p-AMPKα, p-ACC, and Actin. The entire blots are shown in Supplementary Fig. 7. The densitometric values for p-AMPKα were normalized to Actin (bottom). (c and d) BMDMs were transduced with non-specific shRNA (shNS) or Ampk-specific shRNA (shAmpk)-expressing lentivirus for 48 h and then infected with ERFP-Mtb (moi = 10) for 4 h, followed by treatment with OMS-A or OMS-B for 24 h. (c and d) Mtb-ERFP (red), Alexa 488-conjugated-LC3 (green), and DAPI (blue) were detected by confocal analysis. Scale bar, 5 µm. (c) Representative confocal microscopic images from three independent samples are shown. (d) Quantitative analysis of cells showing the colocalization between LC3 and Mtb-ERFP. For each experiment, at least 100 cells were scored from 6 random fields. (e) After 3 days of infection, intracellular bacterial loads were determined by CFU assay. (inset) RT-PCR analysis of Ampk mRNA expression of transduction efficiency. Data shown are from one representative of at least three independent experiments (means ± SD of triplicates [a,b bottom; d] samples]). *p < 0.05, ***p < 0.001, compared with shNS. U, uninfected/untreated; SC, solvent control.