Cell culture
Murine macrophage RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 mg/mL) at 37 °C with 5% CO2. The cells were cultured in six-well polypropylene tissue culture plates in a 5% CO2 atmosphere at 37 °C for 24 h before infection to allow them to adhere.
M. smegmatis culture and intracellular survival assay
Mycobacterium smegmatis (ATCC 700084/mc2 155) was cultured in Middlebrook 7H9 liquid medium supplemented with 10% oleic acid, albumin, dextrose, and catalase, and 5% glycerol. M. smegmatis was harvested by centrifugation (3000 rpm, 30 min), washed, and resuspended in phosphate-buffered saline (PBS) to a concentration of 5 × 107 CFU/mL. The bacterial cells were stored at − 80 °C until used. Heat-killed M. smegmatis was prepared by heating of live M. smegmatis in PBS at 80 °C for 30 min.
Cells were infected with M. smegmatis at an MOI of 5 and incubated for 1 h at 37 °C in a 5% CO2 atmosphere. After allowing time for phagocytosis, the cells were washed with PBS to remove extracellular bacteria, and incubated with fresh medium without antibiotics for a further. To assay intracellular survival, M. smegmatis-infected cells were lysed in sterile distilled water and disintegrated in a water bath sonicator for 3 min to collect intracellular bacteria. The lysates were plated separately on Middlebrook 7H10 agar plates and incubated for 14–21 days. Colony counts were performed in triplicate.
Chemicals and antibodies
Chemicals and inhibitors were dissolved in dimethyl sulfoxide and diluted to the desired concentrations directly in the culture medium. RAW 264.7 cells were pretreated with the indicated concentrations of inhibitors for 1 h before M. smegmatis infection. TM, z-LEHD-fmk (caspase-9 inhibitor), z-VAD-fmk (pan-caspase inhibitor), SP600125 (p-JNK inhibitor), PD98059 (p-ERK inhibitor), SB203580 (p-p38 inhibitor), BAY 11-7082 (NF-κB inhibitor), and caffeic acid phenethyl ester (NF-κB inhibitor) were purchased from Calbiochem (San Diego, CA, USA). NAC, cytochalasin D, staurosporine, and 4-PBA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Z-ATAD-fmk (caspase-12 inhibitor) was purchased from R&D Systems (Minneapolis, MN, USA). Lipopolysaccharide and H2O2 were purchased from InvivoGen (San Diego, CA, USA).
Western blotting was performed using antibodies against caspase-12, caspase-9, caspase-3, PDI, ERO1α, p-protein kinase RNA-like ERK, p-eIF2α, CHOP, Bip, p-p38, p-JNK, p-ERK, p-IκBα, IκBα, JNK, ERK, p38 (Cell Signaling, Danvers, MA, USA), and ATF6α (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Goat anti-rabbit IgG (Cell Signaling) and goat anti-mouse IgG (Calbiochem) were used as secondary antibodies. β-actin (Santa Cruz Biotechnology) was used as a loading control.
Western blot analysis
Whole cells were lysed in radio-immunoprecipitation assay buffer (ELPIS Biotech, Daejeon, Korea) containing a protease inhibitor cocktail. Extracted proteins were separated on 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked with Tris-buffered saline, 0.1% Tween 20 (TBS-T) containing 5% skim milk (Santa Cruz Biotechnology) at room temperature for 1 h and then incubated with primary antibodies (1:1000) overnight at 4 °C. After washing with TBS-T, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000) for 2 h at room temperature. The bound antibodies were detected using a chemiluminescent HRP substrate (Enhanced chemiluminescence (ECL); Millipore, Billerica, MA, USA). Band intensities were quantitated using Omega Lum C (Aplegen Inc., Pleasanton, CA, USA).
Apoptosis analysis
Apoptosis was assessed using an Annexin-V-PI staining kit according to the manufacturer’s instructions (BD Pharmingen, San Diego, CA, USA). Cells were analyzed using a fluorescence-activated cell sorting (FACS) Canto II flow cytometer (BD Biosciences, San Jose, CA, USA) and the data were processed using Flow Jo software (Tree Star, Ashland, OR, USA). Staurosporine (500 nM, 6 h) was used as the positive control.
Measurement of ROS levels
RAW 264.7 cells were harvested using PBS. For ROS staining, the cells were incubated with dichlorofluorescein diacetate (DCFH-DA; 5 μM; Molecular Probes, Eugene, OR, USA) or dihydroethidium (DHE; 2 μM; Molecular Probes) for 30 min in a 5% CO2 atmosphere at 37 °C. RAW 264.7 cells were washed three times with PBS and fixed with 4% paraformaldehyde. After being washed with PBS, DCFH-DA-positive cells were analyzed in a FACS Canto II cytometer (BD Biosciences) and the data were processed using Flow Jo software (Tree Star). DHE staining was analyzed using an optical microscope (Olympus BX51, Olympus, Japan). H2O2 was used as the positive control.
Reverse transcription polymerase chain reaction
Total RNA was extracted from RAW 264.7 cells infected with M. smegmatis using TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The extracted mRNA was transcribed into cDNA using a reverse transcription kit (ELPIS Biotech). cDNA was amplified using Prime Taq Premix (Genet Bio, Daejeon, Korea) to assess XBP-1 splice, CHOP, and Bip mRNA levels. β-actin was used as the control.
Enzyme-linked immunosorbent assay
A sandwich enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences) was used to quantify TNF-α, MCP-1, and IL-6 levels in the culture supernatants of M. smegmatis-infected RAW 264.7 cells. Assays were performed following the manufacturer’s instructions. Triplicate samples were analyzed using an ELISA reader and compared with a standard curve.
Statistical analyses
All experiments were performed in at least triplicate, and representative results are presented. Statistical significance was analyzed using Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA) by one-way analysis of variance. The data are expressed as means ± standard deviations. Statistical significance was set to p < 0.05, p < 0.01, and p < 0.001.