Primary bovine alveolar macrophages and RAW264.7 cells
This study was approved by the ethics committee for use and care of animals at Ningxia University (Yinchuan, China). Bovine alveolar macrophages (AMs) were obtained from lungs of one- to two-year-old Simendal cattles from a tuberculosis-free herd. The entire lung was sterilely removed post-mortem with a portion of trachea, and was intratracheally infused with 500 mL of D-Hank's solution (Biotopped, Beijing, China) containing 50μg/mL of gentamicin, 2.5μg/mL of amphotericin and 100μg/mL of penicillin and streptomycin (Hyclone, Logan, USA). The recovered bronchoalveolar lavage fluid (BALF) was filtered by passing a 70 µm-pore nylon cell strainer prior to being collected into sterile beakers and centrifuged at 1000 rpm for 10min. The cell pellet was then resuspended in 20 mL RPMI-1640 medium (HyClone, Logan, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, USA), 100 U/mL of penicillin and 100 U/mL of streptomycin. 3 mL of red blood cell (RBC) lysate solution was then added into the cell suspension and gently mixed well for 3min at room temperature (RT) before the cells were recollected by centrifugation at 1000 rpm for 10min. The cells were resuspended with RPMI-1640 medium with 10% FBS and counted. The resultant primary cells were seeded at a density of 5´107 per tissue culture dish with diameter of 140 mm and cultured in RPMI-1640 medium with 10% FBS for 6 h to 8 h. The unattached cells were removed by rinsing the culture with pre-warmed PBS, and the attached monolayer of primary AMs were dissociated with TrypleTM Express (Thermo Fisher Scientific, Shanghai, China) and harvested by centrifugation. The isolated cells were then re-plated in 6-well plates at a density of 5×106/well in RPMI-1640 medium with 10% FBS and cultured for 16-18 hours for subsequent experiments. The murine macrophage cell line RAW264.7 was purchased from Shanghai Academy of Life Sciences, Chinese Academy of Sciences (Shanghai, China). Cells were maintained in DMEM medium supplemented with 10% FBS and penicillin/streptomycin. All cells were cultured in humidified incubators with 5% CO2 atmosphere at 37°C.
BCG culture and infection
The M. bovis BCG vaccine strain was purchased from Chengdu Institute of Biological products (Chengdu, China). BCG bacterial cells were grown in the Middlebrook 7H9 medium (BD Difco, San Jose, CA, USA) supplemented with 10% albumin-dextrose-catalase (ADC) enrichment medium (BD Difco, San Jose, CA, USA) and 0.05% Tween 80 (Sigma, St. Louis, MO, USA) at 37°C with slow shaking for 2 weeks. The bacteria cells were harvested by centrifugation and re-suspended in the culture medium. The bacterial cell number was titrated by spectrophotometer at wavelength of 600 nm based on an OD600nm of 1.0 equivalent to 1×108 mycobacterial cells . The bacteria stocks were aliquot and stored at −80 °C freezer for subsequent uses. For infection, the macrophage cells were cultured with 6-well plates for 6-8h before they were infected with BCG at a multiplicity of infection (MOI) of 10 bacteria and then incubated for additionally various times.
The total RNA of cells was isolated using Trizol reagent per manufacturer’s instruction (Invitrogen, Grand Island, NY, USA). The reverse transcription of ﬁrst-strand cDNA synthesis was generated using M-MLV reverse transcriptase (TaKaRa, Dalian, China). The quantitative reverse transcription PCR (qRT-PCR) was performed in the Roche Lightcycler 2.0 using TaKaRa SYBR Green I kit (Takara, Dalian, China). The primer sets used for RT-PCR were designed and synthesized in Shanghai Sangon Biotech Inc. (Shanghai, China) by bioinformatics tools using available mRNA sequences (Suppl. Table 1). The relative expression of genes of interest was calculated by accessing the efﬁciencies and the crossing point deviation of a given gene vs housekeeping β-actin gene. In each independent experiment, The relative changes of gene expression were represented by folds of change over its respective uninfected control cultures by a 2-ΔΔCT method.
Whole cell extracts were prepared by lysing cell cultures in lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40) for 60 min on ice. The concentration of soluble protein was determined with Bio-Rad Protein Assay based on the method of Bradford (Bio-Rad Laboratories, Richmond, CA, USA). The clarified lysates (100 µg) were resolved in 8% or 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel (SDS-PAGE) and then transferred to nitrocellulose membranes for immunoblotting assay probed with antibodies to proteins of interest. The primary antibodies used in this study were listed in Suppl. Table 2. All these primary antibodies were applied in a dilution of 1:500-1000. Following extensively washing, protein of interest was detected or visualized with an appropriate HRP-labelled or fluorescence-labelled IRDye (Li-Cor Biosciences, Lincoln, NE, USA) secondary antibody. The blots were then developed using the enhanced Western Bright ECL reagent (Advansta, Menlo Park, CA, United States) or Li-Cor Odassay Scanner (Li-Cor Biosciences). The relative expression of protein was semi-quantified by optical densitometry using ImageJ Software version 1.46 (http://rsb.info.nih.gov/ij/). The densitometric arbitrary unit (A.U.) was used for determined the ratio between the net intensity by calculating values of each sample divided by the β-actin internal control.
Determination of cholesterol and cholesterol ester
The contents of intracellular cholesterol and cholesterol ester were determined by commercially available cholesterol quantitative assay kit according to manufacturer's instructions (Sigma, St. Louis, MO, USA). The absorbance was then detected at 570nm. Finally, the contents of intracellular total cholesterol and free cholesterol were calculated comparing to its standard curve, and the amount of cholesterol ester was calculated by the substrate of the content of total cholesterol to the content of free cholesterol.
Generation of RAW264.7 cell lines overexpressing or silencing ACAT1
To generate a lentiviral vector overexpressing ACAT1, cDNA of murine acat1 gene (NM_144784) was cloned downstream of the CMV promoter of GV492 lentiviral proviral backbone plasmid (Genechem Co., Ltd., Shanghai, China); to generate lentiviral vectors knocking down endogenous ACAT1 expression, shRNAs targeting sequences of 5’TCGGTCTGGCTAGTATTTG3’, 5’CGTACCTAAGGTTCTTAAA3’ and 5’TAACTGATGTCTACAATAA3’ of murine acat1 gene (NM_144784) were respectively cloned downstream of the U6 promoter of GV493 lentiviral proviral backbone plasmid (Genechem Co.,Ltd., Shanghai, China). The constructed proviral plasmids were used for generation of respective VSV-G pseudotyped lentiviral vectors Lenti-ACAT1 and Lenti-shRNA-ACAT1 as described elsewhere. To generate murine macrophage RAW264.7 cells overexpressing and silencing ACAT1, RAW264.7 cells were infected with Lenti-ACAT1 and Lenti-shRNA-ACAT1, respectively. The virally transduced cells were then cultured for 72 h following the infection before they were refreshed with a selective medium containing purinomycin for additional 4-5 days. The cell pools with were then used for further experiments after functional determination.
Suppression of of ACAT1 using inhibitor K604
RAW264.7 cells were seeded in a 6-well plate culture dish at a density of 2×106/well in DMEM-10% FBS culture medium containing 10 μM of ACAT1 protein inhibitor K604 (MedChemExpress, USA) and BCG at a multiplicity of infection of 10:1. The cells were and cultured for 12 h before they were used for analysis.
2.8 Statistical analysis.
All data collected in this study were from at least three independent biological repeated experiments, which were analysed using SPSS statistics 22.0 (SPSS Inc., Chicago, IL, USA). The data was presented as the mean ± SD. Statistical differences between groups were analysed by one-way analysis of variance (ANOVA), followed by post-hoc Tu-key’s test. The data was presented as the mean ± SD. A P values < 0.05 were considered as statistically significant.