Cell culture, transient transfection, generation of stable cell clones

COS1 is a fibroblast-like cell line derived from monkey kidney. HEK 293 T cells are human embryonic kidney cells containing SV40 T-antigen. These cells were grown in DMEM supplemented with 10% FBS and 1% penicillin streptomycin in a 5% CO2 incubator at 37 °C. For transfection, 15 µg of plasmids and 45 µL transfection reagent were mixed in 1.5 mL serum-free media before they were added to cells in a 10-cm dish at 60% confluency. Cells were harvested after incubation for 48 h.

We used a tetracycline-inducible (Tet-On) lentiviral system to express exogenous CYP11A1 only when cells were treated with tetracycline or its analog, doxycycline. To generate cell clones for inducible CYP11A1 expression, COS1 cells were co-transduced with two lentiviruses containing CYP11A1-HF-IRES-EGFP and rtTA3 cDNAs in the presence of 8 μg/mL polybrene. Infected cells were selected with 1 μg/mL puromycin for 2 weeks. To isolate cell clones bearing CYP11A1-HF-IRES-EGFP cassette, the puromycin-resistant (rtTA3-positive) clonal cells were treated with 1 μg/mL doxycycline and examined for EGFP signal in a fluorescent microscope. EGFP-positive clones were isolated and CYP11A1 expression was further confirmed by immunoblotting with anti-Flag antibody 24 h post-induction with doxycycline. Two independent cell clones, C1 and C4, were selected and used for later experiments.

Reagents, RNA, plasmids, and cloning

The reagents used here such as antibodies, oligonucleotides, plasmids, enzymes, kits, and software are listed in Additional file 1: Table S1.

For RNA isolation, about 50 mg of 3-month-old zebrafish testis tissue samples were homogenized in 0.5 mL Trizol (Ambion). RNA was extracted by chloroform, precipitated in isopropanol, and dissolved in 50 µL diethyl pyrocarbonate-treated water.

For the cloning of zebrafish cyp11a1 and cyp11a2 cDNA, zebrafish testis RNA was used as a template for cDNA synthesis with Maxima Reverse Transcriptase and primers specific for cyp11a1 and cyp11a2. Human CYP11A1 (AA #1–521) was reported before [25]. CYP11A1 and all the cDNA fragments (AA #1–39 and AA #1–85) were cloned into the XhoI and EcoRI sites of pEGFP-N1 vector, or the AflII and BamHI sites of pcDNA3-EGFP-APEX2 vector. The A’-helix of human CYP11A1 (AA #57–68) was subcloned into the EcoRI and BamHI sites of pCYP11A1(39)-EGFP vector, or pcDNA3-CYP11A1(39)-EGFP-APEX2 vector. The resulting constructs (pCYP11A1(39)-EGFP, pCYP11A1(39 + A’)-EGFP, pCYP11A1(85)-EGFP, pCYP11A1(521)-EGFP, pcDNA3-CYP11A1(39)-EGFP-APEX2, pcDNA3-CYP11A1(39 + A’)-EGFP-APEX2, pcDNA3-CYP11A1(85)-EGFP-APEX2, and pcDNA3-CYP11A1(521)-EGFP-APEX2) were further validated by DNA sequencing.

For the generation of lentiviral construct that expresses CYP11A1, full-length CYP11A1 cDNA was inserted into the Eco RI and Bam HI sites of a Tet-On lentiviral vector PL-SIN-5TO-HF-IRES-EGFP [26], which contains a C-terminal HA-FLAG pPAX2 (HF) tandem tag and an IRES-EGFP cassette driven by five repeats of Tet operators and a mini-CMV promoter. The resulting plasmid, PL-SIN-5TO-CYP11A1-HF-IRES-EGFP, was validated by DNA sequencing. The pTRIPZ-rtTA3 plasmid harbors a coding sequence for the reverse tetracycline-transactivator 3 (rtTA3) and an IRES-puromycin cassette under the control of an EF1a promoter [26]. The pPAX2 and pMD2.G plasmids for lentiviral packaging were purchased from Addgene.

The lentivirus expressing PL-SIN-5TO-CYP11A1-HF-IRES-EGFP or pTRIPZ-rtTA3 were prepared according to a lentivirus-packaging protocol from Addgene. Briefly, 1 μg lentiviral plasmid, 0.75 μg pPAX2, and 0.25 μg pMD2.G were co-transfected into HEK 293 T cells with 6 µL TransIT-LT1 transfection reagent (Mirus Bio). Lentivirus-containing supernatants were collected 48 h post-transfection.

Hsp60 knockdown

Hsp60 was knocked down by si-HSP60 RNA (Dharmacon) following manufacturer’s instructions. Briefly, cells were transfected with 25 nM of si-HSP60 RNA for 24 h followed by addition of 1 µg/mL doxycycline and incubation for 24 h to induce exogenous CYP11A1 expression. Cells were transfected again with si-HSP60 RNA and incubated for another 24 h to ensure adequate depletion of Hsp60 before harvesting.

Immunofluorescence

Cells grown on coverslips inside a 12-well plate were washed with phosphate-buffered saline (PBS) pH 7.4 and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Following three PBS washes, cells were permeabilized in 0.2% Triton X-100 (in PBS) for 10 min. After three PBS washes, the coverslip was blocked with 10% normal goat serum in PBS for 1 h at room temperature. The primary antibody was added to the plate after removal of the blocking solution and incubated overnight at 4 °C. After three PBS washes, the coverslips were incubated with secondary antibody (Alexa Fluor 546 Thermo) and 10 µg/mL DAPI for 1 h at room temperature, washed with PBS three times and mounted on a microscope glass slide. Slides were dried in the dark overnight. Images were acquired using a Zeiss LSM710 inverted confocal microscope and processed using ZEN 2011 (Blue edition) software.

Protein extraction, membrane protein extraction

Proteins were extracted from cells after homogenization in 2 mL lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% digitonin, 1 × protease inhibitor) using an electric homogenizer with an adapter pestle. Cell lysates were incubated on ice for 30 min before centrifugation for 17,000×g for one min at 4 °C to remove unbroken cells and debris. Proteins in the supernatant were quantified by Bradford Assay and used for succeeding experiments.

For membrane protein extraction, cells were homogenized in 1 mL of 0.1 M Na2CO3 pH 11.5 using an electric homogenizer with an adapter pestle followed by incubation on ice for 30 min. The homogenates were centrifuged at 435,400×g for 1 h using a Beckman rotor TLA 120.1. Pellet fractions were resuspended in alkaline buffer to achieve homogeneity. All fractions were kept in -80 °C until further processing.

Identification of CYP11A1-interacting proteins

To identify CYP11A1-interacting proteins, about 4 mg of cell lysate from stable clones C1 and C4 in 500 µL TBS (50 mM Tris pH 7.4, 150 mM NaCl) was incubated with 100 µL anti-FLAG M2 or anti-HA affinity bead slurry (Sigma Aldrich) with gentle shaking at 4 °C overnight. After centrifugation at 8200×g for 1 min, beads were washed with 500 µL TBS three times. Proteins were eluted by incubating the beads in 200 ng/µL of 3X FLAG (Sigma Aldrich) or 1X HA peptide (Sigma Aldrich) with gentle rotation at 4 °C for 1 h followed by centrifugation at 8200×g for 1 min. Samples were separated on SDS-PAGE for Western blot analysis.

Isolation of mitochondria

Mitochondria were isolated from cells using Mitochondria Isolation Kit for Mammalian Cells (Thermo Scientific). Briefly, about 4 × 107 cells were lysed and cell nuclei removed by centrifugation at 700×g for 10 min at 4 °C. Supernatant was collected and centrifuged at 12,000×g for 15 min at 4 °C. Mitochondria in the pellet fraction was collected, washed, and centrifuged again at 12,000×g for 5 min at 4 °C. Pelleted mitochondria were solubilized in sample buffer (50 mM Bis–Tris pH 7.2, 50 mM NaCl, 10% w/v glycerol, 0.001% Ponceau S).

Transmission electron microscopy (TEM)

Cells grown in a 12-well cell culture plate lined with Aclar film (Electron Microscopy Sciences) were fixed with 2.5% glutaraldehyde for 20 min and washed with 0.1 M cacodylate buffer pH 7.2. Cells were then post-fixed with 1% osmium for 30 min, washed with distilled water three times, fixed again in 1% uranyl acetate for 30 min, and washed again with distilled water three times. Cells were then sequentially dehydrated in 50%, 70%, 95% and 100% ethanol for 5 min each and washed three times with distilled water. The film was mounted in rubber mold with Epon resin and incubated at 65 °C for 48 h. Resin blocks were cut into 70 nm thickness by a diamond knife on ultramicrotome (Leica EM UC7). Cell slices were placed on mesh grid then post-stained with 4% uranyl acetate for 3 min and lead citrate for 10 min. Ultrathin sections were viewed on Tecnai G2 Spirit TWIN (Thermo) transmission electron microscope operated at 120 kV. Images were acquired via GATAN CCD SC1000 (4008 X 2672 active pixels) camera and processed by Gatan Digital Micrograph software.

Enzymatic activity by ELISA assay

To examine the activity of CYP11A1, stable clones C1, C4, and the controls were incubated with 12.5 µM 22-hydroxycholesterol overnight. P5 was detected using an ELISA kit (LDN). Briefly, about 50 µL of cell culture media were pipetted into a microwell plate coated with rabbit anti-P5 antibody followed by the addition of 100 µL of P5-horseradish peroxidase (HRP) conjugate working solution. After incubation for 1 h followed by three washes, 150 µL tetramethylbenzidine and hydrogen peroxide substrate were added to the plate and incubated for another 15 min. The reaction was stopped with the addition of 50 µL 1 M sulfuric acid. The amount of P5 was measured by an Emax Precision Microplate Reader (Molecular Devices) and analyzed by Softmax Pro 5.3.

Mass spectrometry (MS) analysis

Samples obtained from affinity chromatography were analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS for protein identification. Both methods of in-gel and in-solution trypsin digestion were used. For in-gel digestion, bands in Coomassie-stained gel were excised, cut into small pieces, and washed twice with 25 mM NH4HCO3 in 40% methanol while vortexing for 10 min. The samples were dehydrated with 100% acetonitrile before drying in a vacuum centrifuge. For in-solution digestion, about 4 µg proteins were lyophilized in a vacuum centrifuge for 2–3 h.

Protein samples were redissolved in 30 µL distilled water, and reduced by incubation in 5 mM dithiothreitol, 8 M urea, 50 mM ammonium bicarbonate pH 7.8 at 37 °C for 1 h. Samples were then alkylated with 15 mM iodoacetamide for 30 min in the dark at room temperature before digestion with 12.5 ng/μL sequencing grade modified trypsin in 25 mM ammonium bicarbonate containing 10% v/v acetonitrile for 12–16 h at 37 °C. The reaction was stopped through the addition of formic acid to 5% [27]. After salt removal with Millipore C18 Zip-tip, the peptide solution was dried by vacuum centrifugation before dissolution in 30 µL distilled water.

The mass spectra were acquired using two different machines. First spectra were acquired on a Bruker New UltrafleXtremeTM mass spectrometer equipped with an Nd-YAG laser (255 nm) operating at a rate of 200 Hz. One µL of the protein solution was mixed with 1 µL of matrix (10 mg/mL alpha-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid) directly on a stainless steel MALDI plate and dried. Spectra were recorded in a reflector positive ion mode using an accelerating voltage of 20 kV. The instrument was calibrated with known standards (ovalbumin, serum albumin, myoglobin, cytochrome c, β-lactoglobulin, and Angiotensin 1) to obtain an accuracy of 5 ppm. The mass spectrum was obtained by averaging 2000 laser shots, and the data were processed and analyzed using Flex Analysis software 3.4 (Bruker, Daltonics).

The second MALDI-TOF MS analysis was performed in positive ion mode with delayed extraction (reflection mode) on a Bruker Autoflex III MALDI TOF/TOF mass spectrometer equipped with a 200 Hz SmartBean Laser. About 0.5 μL of the supernatant from the digest was rigorously mixed with 0.5 μL matrix solution (5 mg/ml dihydrobenzoic acid in 0.1%TFA and 30% acetonitrile), and 0.3 μL aliquots of each resulting mixtures were deposited onto the 384/600-μm MTP AnchorChip (Bruker Daltonics). Data were acquired using FlexControl 3.4 and processed by Flex-Analysis 3.4 (Bruker Daltonics). The data were further processed via Biotools 3.2 (Bruker) package accessing the online Mascot server (www.matrixscience.com) to identify corresponding polypeptides against the Swiss-Prot or NCBI database. The parameters for database searches were set as follows: carboxyamidomethylation on cysteine (fixed modification), oxidation of methionine (variable modification), 60 ppm of peptide mass tolerance, 0.7 Da of fragment mass tolerance and 2 missed cleavages. The representative mass spectra are shown in Additional file 1: Fig. S1.

Polyacrylamide gel electrophoresis and Western blot

Thirty µg of proteins were loaded on 12% SDS polyacrylamide gel before electrophoresis at 90 V for 15 min followed by 130 V for 2.5 h until the protein ladder was visibly separated. For blue native gel electrophoresis (BN-PAGE), about 50 µg mitochondrial protein was loaded in 3–12% Bis–Tris Gel (Novex). Initial run was set at 150 V for 45 min with 200 mL dark Coomassie blue cathode buffer pH 6.8 (50 mM Bis–Tris, 50 mM Tricine, 10 mL 5% G-250 Coomassie blue) and 600 mL anode buffer pH 6.8 (50 mM Bis–Tris, 50 mM Tricine). After the initial run, the cathode buffer was replaced with 200 mL light Coomassie blue buffer pH 6.8 (50 mM Bis–Tris, 50 mM Tricine, 1 mL 5% G-250 Coomassie blue) and voltage was increased to 250 V for 1 h.

Proteins were transferred on PVDF membrane in 1-L transfer buffer (48 mM Tris, 39 mM glycine, 0.037% SDS, 20% methanol) at 80 V for 2 h. For blue native gels, proteins on the blot were additionally fixed with 8% acetic acid for 15 min.

Blots were blocked in 5% milk in PBS-T pH 7.4 (0.1% Tween20 in PBS) followed by incubation with primary antibodies overnight at 4 °C with slow shaking. After three PBS-T washes, secondary antibody was added and incubated for 1.5 h at room temperature with slow shaking followed by three PBS-T washes. The immunoreactive signals were visualized using an enhanced chemiluminescence substrate in a bio-imaging system (UVP Biospectrum 815). The original uncropped gel pictures are shown in Additional file 1: Figs. S2–S9.

Oxygen consumption measurement

Oxygen consumption rates of cultured cells were measured using a Seahorse XF Cell Mito stress test kit (Agilent). About 40,000 cells were seeded in a 24-well XF24 microplate (Agilent) and grown in DMEM with or without doxycycline for 6 h. After the monolayer cell culture reached about 90% confluency, cell media were replaced with Seahorse XF DMEM media freshly supplemented with 1 mM sodium pyruvate, 2 mM glutamine and 10 mM glucose and incubated at 37 °C without CO2 for 1 h before oxygen consumption was measured using an Agilent Seahorse Analyzer. Cells were treated sequentially with 0.5 µM oligomycin, 2 µM carbonyl cyanide-4-trifluoromethoxyphenylhydrazone, and 0.5 µM rotenone/antimycin. Data were analyzed using Wave Software and Seahorse XF Cell Mito Stress Test Report Generator.

Quantification and statistical analysis

Western blot results were quantified using Image J software. For cristae structure quantification, more than 50 cells were counted from each sample. Data are shown as mean with standard deviation (SD). Unpaired non-parametric t-test was used for statistical analysis. N represents the number of independent experiments. For correlating the amounts of CYP11A1 and MIC10 complexes, linear regression was obtained by plotting the data and analyzed using non-linear fit of XY correlation. All statistical analyses were performed using GraphPad Prism8.