The J774A.1 murine macrophage cell line was purchased from the China Center for Type Culture Collection (Nanjing, China), and maintained in an atmosphere of 5% CO₂ at 37°C in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal calf serum (FCS). The rho0 cell line (J774A.1 rho0) was generated by incubation of J774A.1 in DMEM containing 10% FCS, 1 mM pyruvate, 50 µg/ml uridine and 50 ng/ml ethidium bromide (EB), all from Sigma-Aldrich (St. Louis, MO, USA), for 90 days at 37°C, as described previously (14). Rho0 cell auxotrophy for uridine and pyruvate was assessed by performance of a Cell Counting Kit-8 assay (Guangzhou Yiyuan Biotech Co. Ltd, Guangzhou, China) to confirm the impairment of growth in the absence of these nutrients.

mtDNA from J774A.1 cells (China Center for Type Culture Collection, Wuhan, China) was semi-quantified by PCR to evaluate the remaining mtDNA in the rho0 cells. Total DNA was isolated using DNAzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and semi-quantified by PCR was performed on a thermal cycler (2720; Thermo Fisher Scientific, Inc.) using PCR Master Mix (2X) (Thermo Fisher Scientific, Inc.) in a final volume of 50 µl. The conditions for PCR were as follows: 94°C for 10 sec, 25 cycles at 94°C for 45 sec, 55°C for 60 sec and 72°C for 45 sec. The primer sequences used for PCR were as follows: mtCOI, F 5′-GCCCCAGATATAGCATTCCC-3′, R 5′-GTTCATCCTGTTCCTGCTCC-3′. 18S rRNA, F 5′-TAGAGGGACAAGTGGCGTTC-3′, R 5′-CGCTGAGCCAGTCAGTGT-3′. All primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). 18s rRNA was used as a refernce gene. The clones containing less mtDNA were then selected and cultured for 12 weeks in the aforementioned medium, but without ethidium bromide.

J774A.1 normal and rho0 cells were plated in 6-well plates and grown to 50~60% confluence. Following stimulation with 100 µl Ox-LDL, the cells were harvested and lysed using 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich). The cell lysates were centrifuged at 14,000 × g for 30 min at 4°C. Then the protein concentrations in the supernatants were determined using a bicinchoninic acid kit (Beyotime Institute of Biotechnology, Haimen, China). Proteins (20 mg) from the homogenized samples were separated by 15% SDS-PAGE for 1 h at 200 V and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Boston, MA, USA). After blocking with Tris-buffered saline and Tween-20 (TBST) containing 5% non-fat milk powder for 2 h at room temperature, membranes were treated with the following primary antibodies: Rabbit polyclonal anti-pro-caspase 1 (1:500; cat. no. BS1730; Bioworld Technology, Inc., St Louis Park, MN, USA); rabbit polyclonal anti-cleaved caspase 1 (1:500; cat. no. BS1730; Bioworld Technology, Inc.); rabbit polyclonal anti-IL-1β (1:500; cat. no. BS6067; Bioworld Technology, Inc.) and rabbit polyclonal anti-β-actin (1:1,000; cat. no. BS-0061R; BIOSS, Beijing, China) at 4°C overnight. Next, they were washed with TBST three times and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:10,000; cat. no. A0208; Beyotime Institute of Biotechnology) at room temperature. Immunoreactivities were detected using an enhanced chemiluminescence detection system (Fast Western Blot kit, ECL substrate; Thermo Fisher Scientific, Inc.) and images were obtained using an automatic digital gel image analysis system (4500SF; Tanon Science & Technology Co., Ltd., Shanghai, China).

J774A.1 normal and J774A.1 rho0 cells were seeded in 6-well plates and grown to 50~60% confluence. 0, 25, 50 and 100 µg/ml Ox-LDL was added to the supernatants for 24 h. The cells were washed with phosphate-buffered saline (PBS) and fixed with formaldehyde. The fixed cells were stained with Oil Red O dye (Nanjing Jiancheng Biotechnology Institute Co., Ltd., Nanjing, China) for 30 min at 37°C. The status of intracellular lipid accumulation was detected by microscopy (Nikon Eclipse 80i; Nikon Corporation, Tokyo, Japan) to assess lipid aggregation.

Following stimulation with Ox-LDL, cells were harvested and sonicated on ice for 5 sec, with 15 sec intervals using an ultrasonic homogenizer (JY88-IIN; Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China). Following sonication for 10 min, the cells were centrifuged at 8,000 × g for 10 min at 4°C. The supernatants were then collected and used for total cholesterol quantification using the CHOD-PAP method and a Total Cholesterol Assay kit (Nanjing Jiancheng Biotechnology Institute Co., Ltd.), according to the manufacturer's instructions.

Cell death was assessed by lactate dehydrogenase (LDH) release (Beyotime Institute of Biotechnology), and EB and acridine orange (AO) staining assays (Sigma-Aldrich).

LDH is a cytosolic enzyme that is released from damaged cells, the presence of which can be detected by the conversion of lactate to pyruvate with a parallel reduction of nicotinamide adenine dinucleotide (NAD) to NADH. For LDH release, 50 µl supernatants of J774A.1 normal and rho0 cells following treatment with Ox-LDL were collected and measured using the LDH Cytotoxicity Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions.

EB in combination with AO (EB/AO) is widely used to evaluate cell death (15). While AO is acidophilic green dye that preferentially stains the lysosomes and nuclei of live cells, EB emits a red fluorescence upon binding to DNA. For EB/AO staining (Sigma-Aldrich), suspended and adherent cells in 96-well plates were collected by centrifugation at 400 × g for 5 min and dyed with 8 µl EB/AO mix per well (50 µg/ml v/v). Experiments were performed in triplicate and images were acquired using an inverted fluorescence microscopy (Nikon Eclipse 80i; magnification, ×400).

The quantity of ROS generated in 100 µg/ml Ox-LDL-treated J774A.1 normal and rho0 cells for 3 h was measured by fluorescence-activated cell sorting (BD FACSVerse; BD Biosciences, Franklin Lakes, NJ, USA) using fluorescent 2′,7′-dichlorodihy-drofluorescein diacetate (DCFH-DA; Sigma-Aldrich).

Following treatment with 100 µg/ml Ox-LDL for 24 h, J774A.1 normal and rho0 cells were triple-stained with MitoTracker Deep Red, MitoTracker Green and DAPI probes. The change in mitochondrial membrane potential was detected by observing the red signal under a confocal laser scanning microscope (Leica TCS SP5; Leica, Mannheim, Germany).

Data are presented as means ± standard deviation and were analyzed by SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). Statistical analysis was performed using Student's t test for comparison between two groups and P<0.05 was considered to indicate a statistically significant difference.

Ox-LDLs have been previously shown to trigger caspase-1 activation in human macrophages (2). The effect of mtDNA depletion on Ox-LDL-induced caspase-1 activation was examined in the present study in murine macrophages. Cleaved caspase-1, which is proteolytically active, promotes the expression of proinflammatory cytokines, such as IL-1β and IL-18 (4). It was found that Ox-LDL stimulation did not increase the expression levels of cleaved caspase-1 and IL-1β in rho0 cells. As shown in Fig. 1, cleaved caspase-1 and IL-1β expression levels were markedly reduced in J774A.1 rho0 cells when compared with the J774A.1 normal cell group following treatment with 100 µg/ml Ox-LDL for 24 h.

Rho0 cells have been reported to resist apoptosis (12). Whether mtDNA-depleted cells are resistant to Ox-LDL-induced macrophage pyroptosis was investigated in the present study. As hypothesized, it was found that mtDNA-depleted J774A.1 (rho0) cells exhibited a decreased ratio of dead cells when compared with J774A.1 normal cells following treatment with 50 and 100 µg/ml Ox-LDL for 24 h (Fig. 2A–F). Consistent with the results from EB/AO staining, a significant reduction in LDH release was observed in the rho0 cells when compared with the normal J774A.1 cells following treatment with 50 and 100 µg/ml Ox-LDL for 24 h (Fig. 2G). Together, these results indicate that mtDNA-depleted J774A.1 cells are resistant to Ox-LDL-induced cell death.

To investigate whether J774A.1 rho0 cell resistance to ox-LDL-induced cell death was caused by disruption of intracellular cholesterol accumulation, lipid droplets (the existing form of cholesterol in cells) were evaluated by Oil Red O staining. This indicated no observable difference between J774A.1 rho0 and J774A.1 normal cells (Fig. 3). Further quantification of the intracellular cholesterol results using the CHOD-PAP method indicated that mtDNA depletion did not lead to significant disruption in intracellular total cholesterol following treatment with 0, 50 and 100 µg/ml Ox-LDL for 24 h when the J774A.1 rho0 cells were compared with the J774A.1 normal cells. These results indicate that rho0 cells do not affect Ox-LDL-induced cholesterol accumulation in murine macrophages.

The impact of mtDNA depletion on ROS and mitochondrial membrane potential was determined using MitoTracker Deep Red, MitoTracker Green and DAPI triple staining, and H2DCF-DA probes for ROS labeling with a confocal microscope and flow cytometer, respectively. As shown in Fig. 4A, J774A.1 rho0 cells displayed a similar mitochondrial membrane potential to that of J774A.1 normal cells. Following Ox-LDL treatment, a considerable decrement in mitochondrial membrane potential was observed in the J774A.1 normal cells, but not in the J774A.1 rho0 cells. As shown in Fig. 4B, the J774A.1 normal cells displayed a higher ROS level than that of the J774A.1 rho0 cells following Ox-LDL treatment. These results are consistent with the hypothesis that the mitochondrial respiratory chain is the predominant source of Ox-LDL-induced ROS production in J774A.1 cells, and loss of mtDNA enables J774A.1 rho0 cells to maintain the mitochondrial membrane potential.