Reagents and chemicals
SD (purity >95%) was isolated from A. umbellate and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mM (Wang et al. 2008). The attenuated M.tb strain (H37Ra) was purchased from China National Institution For bio-product and drag control. Human monocytic THP-1 cells and A549 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI1640 containing 10% FBS and 1% penicillin/streptomycin mix. Rabbit anti-human VPS34 antibody (ab5451), rabbit anti-human UVRAG antibody (ab174550), rabbit anti-human AMPK antibody (ab131512) and phosphorylated AMPK (ab72845) antibodies were purchased from Abcam (Cambridge, MA, USA). The internal reference beta-actin antibody and secondary antibodies, including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, were purchased from Zhongshan Goldenbridge Biotechnology (Peking, China).
The monocytic cell line THP-1 or A549 was maintained in RPMI 1640 supplemented with 10% foetal calf serum, 2 mM l-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin. Cells were cultured in an incubator at 37 °C with 5% CO2. Before treatments, THP-1 cells were seeded in 6-well culture dishes at a density of approximately 2 × 106/ml in fresh medium. THP-1 cells were then stimulated with 20 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) for 1 day to induce macrophage-like cells.
Cell infection and bacterial enumeration
The attenuated M.tb strain (H37Ra) were used and cultured in Proskauer and Beck (P and B) medium supplemented with 0.05% Tween-80. For visualization of the phagocytosis of M.tb by THP-1 cells, 1 ml of H37Ra bacterial suspension (109/ml) was labelled by incubation with 0.5 mg FITC (Sigma) in 0.1 M carbonate buffer (pH 9.0) at 37 °C for 2 h (Stokes et al. 1993). The bacterial suspension was centrifuged and washed with PBS 3 times to remove unbound FITC. Before infection, the cells were cultured in 6-well plates and washed with RPMI1640 3 times to remove serum and antibiotics. H37Ra bacteria were re-suspended and diluted in antibiotic-free RPMI1640 to infect THP-1-derived macrophages or A549 cells with a multiplicity of infection (MOI) of 5. The infected cells were maintained in an incubator at 37 °C with 5% CO2 for 2 h. The cells were then washed with PBS 3 times to remove the extracellular bacteria. Every 48 h, the culture medium was replaced.
On days 0, 2, 4 and 6, the infected THP-1 or A549cells were lysed with1 ml 0.025% SDS to release the intracellular M.tb. The lysate were then plated as serial dilutions on MB7H11 agar. Colonies were counted after 4 weeks of incubation at 37 °C, and the data are expressed as CFU/ml.
Western blot analysis
The expression of the VPS34, UVRAG, AMPK and phosphorylated AMPK protein in each treatment group was detected by western blotting. Briefly cells were washed with ice-cold PBS 3 times and lysed on ice for 20 min with a western cell lysis buffer (Beyotime, Shanghai, China) containing PMSF, protease inhibitors, and phosphatase inhibitors. The lysis products were then centrifuged at 12,000 rpm for 15 min at 4 °C. Supernatants were standardized for equal protein concentration following the instructions of the BCA Protein Assay Kit (Beyotime, Shanghai, China). The samples were then mixed with loading buffer and boiled in water for 10 min. After separation by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked in 5% (w/v) non-fat dried skim milk powder or 5% BSA (for detecting phosphorylated AMPK)and incubated with diluted primary antibody (1:300 for VPS34, 1:1000 for UVRAG, 1:800 for AMPK, 1:300 for phosphorylated AMPK, 1:200 for beta-actin) overnight at room temperature. After the membranes were washed with PBST 3 times for 15 min, they were incubated with the secondary antibody, goat anti-rabbit IgG (HRP), at a 1:3000 dilution for 2 h. Blots were visualized using enhanced chemiluminescence following the protocol of the manufacturer of the reagent kit (KeyGen, Nanjing, China). The intensity of each band was measured using the Fluor-S MultiImager and QuantityOne software (Bio-Rad, Hercules, CA, USA).
Co-IP was performed following the instructions of the Co-Immunoprecipitation Kit (Thermo Scientific) (Peking, China). Generally, the cells were washed with PBS 3 times and lysed on ice for 30 min using IP lysis buffer (Beyotime) (Beijing, China) containing protease inhibitors (Thermo Scientific) (Peking, China). For removal of the nuclei and intact cells, the lysis products were centrifuged at 20,000×g for 15 min at 4 °C. Protein A agarose was washed and diluted by PBS. The diluted Protein A agarose (50%) was added to the protein at a 1:10 (v/v) ratio. The mixture was incubated on a 4 °C shaking table for 30 min and then centrifuged at 20,000×g for 15 min at 4 °C to collect the supernatant. The supernatants were standardised at 5 μg/μl for equal protein concentration following the instructions in the Bicinchoninic Acid Protein Assay kit (Beyotime, Peking, China). Then, 5 μg rabbit anti-human VPS34 was added to an Eppendorf tube containing 200 μl protein and incubated on a 4 °C shaking table overnight. The antigen–antibody complexes were captured by adding 100 μl Protein A agarose to the samples for 90 min at room temperature. After centrifugation at 20,000×g for 1 min, the Protein A agarose containing the antigen–antibody complexes was washed with ice-cold PBS 3 times. The precipitates were mixed with 5× western blot loading buffer and boiled for 5 min. After centrifugation at 20,000×g for 15 min, the supernatants were prepared for SDS-PAGE.
Analysis of phagosome maturation by confocal microscopy
THP-1-derived macrophages were plated at a density of 2 × 105/ml in 12-well plates on a poly-l-lysine coated glass coverslip in each well. THP-1-derived macrophages were washed 3 times by PBS and infected with 1 × 106/ml FITC-labelled M.tb for 2 h at 37 °C. Cells were then washed 3 times with RPMI1640 to remove extracellular bacteria. After 6 days of culture at 37 °C, the cells were incubated for 20 min in RPMI1640 containing 70 nM LysoTracker Deep Red (ThermoFisher Scientific, ShangHai, China). Then, THP-1-derived macrophages were fixed with 4% paraformaldehyde for 15 min. Slides were then visualized using a confocal microscope (TCS-SP5, Leica, Germany). Co-localization was determined by identifying FITC-linked M.tb (Green) with over lapping LysoTracker(Red). Representative images were obtained with a digital camera and were then processed using Adobe Photoshop 7.0.