Materials
The antibodies were used as follows: anti-AKT (mAb, #4685) and anti-pAkt (mAb, #4060) were purchased from Cell Signaling Technology (Beverly, MA); anti-MYADM (ab72614), anti-Rac1(ab155938), anti-c-Myc (phoshpo S62, ab78318) and MCT-1 (ab135593) were purchased from Abcam (Cambridge, MA); anti-beta-actin (mAb, #66009-1-Ig) was purchased from Proteintech (Proteintech group Inc, Rosement, US). Water-soluble cholesterol was purchased from Sigma Chemical Co. (USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Grand Island, NY, USA).
Cell Culture
The lung adenocarcinoma cells A549 and H1975 were cultured in DMEM medium containing 10% FBS, 100 IU/ml penicillin and 100mg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. For cholesterol (Cho) treatment, A549 and H1975 cells were cultured overnight, and were starved in serum-free DMEM medium for 4 h. The mediaum was replaced by fresh DMEM medium supplemented with 0.8 mg/ml Cho for 72 h.
Clonogenic Assay
A549 and H1975 cells were inoculated in 6-well plates at a density of 800 cells/well, and were treated with 0.8 mg/ml Cho for 14 days. The colone fromation was stained using 1% crystal violet (Sigma) and were captured using a olympus microscope (IX53).
Migration Assay
A549 and H1975 cells were inoculated into the upper chambers containing DMEM complete medium. The bottom chamber was added with DMEM complete medium with 0.8 mg/ml Cho. After 24 h of culture, the cells on the lower surface of the membranes were stained using the crystal violet, followed by analysis using a olympus microscope (IX53).
Invasion Assay
A549 and H1975 cells were plated into the upper chambers coated with Matrigel Matrix (BD, Biosciences, USA). The upper chambers was added DMEM complete medium and the bottom chamber contained DMEM complete medium with 0.8 mg/ml Cho. After 24 h of culture, the cells on the lower surface of the membranes were stained using the crystal violet, followed by analysis using a olympus microscope (IX53).
Gene Transfection
A549 and H1975 cells were inoculated at a density of 1×103 cells/well in 96-well plates. After 12 h of culture, the cells were treated with lentivirus carrying shRNA against MYADM or c-Myc for 48 h, followed by observation by fluorescence microscope. For MYADM overexpression, the cells were transfected with the pcDNA3.1 plasmid carring MYADM ORF using Lipofectamine 3000.
Western Blot
The treated cells was harvested and lysed in RIPA lysis buffer (Thermo). After centrifugation, the protein concentration was determined by BCA method. The equal protein was loaded onto SDS-PAGE, and was transferred onto PVDF membranes. After blocking with 5% fat-free milk, the membranes were incubated with the specified primary antibodies and secondary antibodies. The protein bands were visualized using the enhanced chemiluminescence method and the intensity was analyzed using ImageJ software (RRID:SCR_003070).
Immunofluorescence And Immunohistochemistry
The treated cells were fixed in 4% paraformaldehyde for 20 min and permeabilized using 0.2% Triton X-100 for 5 min. After blocking with 10% goat serum, the cells were labeled with the specified primary antibodies overnight at 4oC, followed by treatment with the corresponding secondary antibody for 1 h at room temperature. After DAPI staining, cell images were obtained using a olympus fluorescence microscope (BX63). For immunohistochemistry assay, frozen sections were fixed in 4% paraformaldehyde for 20 min, and incubated in EDTA solution (pH 9.0) using microwave treatment. After incubating in 3% hydrogen peroxide for 25 min, the sections were blocked using 10% rabbit serum for 30 min, and were then incubated with primary antibodies overnight at 4oCand the HRP-labeled secondary antibodies for 50 min at room temperature. The sections were exposed to fresh DAB solution and counterstained with hematoxylin. The images were captured using a olympus microscope (IX53).
Co-immunoprecipitation
LACs were collected and lyzed in IP lysis buffer for 5 min on ice. After centrifugation, the supernatant was used for co-immunoprecipitation (Co-IP) assay using the Co-IP kit (Thermo Scientific Pierce) according to manufacturer's instructions.
Chromatin Immunoprecipitation (Chip)
The cells were incubated in 1% formaldehyde for 10 min, and were then terminated using glycine solution. The cells were harvested and lysed in SDS lysis buffer, followed by ultrasonication. After centrifugation at 10000 g for 10 min, 100 µl of supernatant were mixed with protein A Agarose for 1 h. After centrifugation, the supernatant was incubated with the specified antibodies overnight at 4 oC. After de-crosslink using NaCl solution, the DNA fragments were purified, and the purified DNA was used as template to amplify target fragment using specific primers.
In Vivo Lac Model
Six-week-old male BALB/c nude mice were obtained from Beijing Vital River Animal Center (Beijing, China) and maintained under specific pathogen-free conditions in the animal center. All procedures were approved by Laboratory Animal Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University. A549 cells or A549 cells carrying shRNA against MYADM were inoculated in nude mice at a density of 2×106 via tail veins. The mice were feed with normal diet or high cholesterol diet (97.5% normal diet, 2% cholesterol and 0.5% sodium cholate) for three weeks, respectively. Tumor proliferation and metastasis were monitored using in vivo imaging system ( IVIS Lumina XRMS Series Ⅲ, PerkinElmer). Subsequently, mice were sacrificed and lung tissues were analyzed using for HE staining and immunohistochemistry assay.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0 software. All data represented the mean ± SD of at least three independent experiments. Difference between two groups was assessed using the Student’s t-test. P < 0.05 was considered statistically significant.