Cell lines and plasmids
The Lung cancer cell lines A549 and H1975 were obtained from the Cell Bank of Shanghai Institutes for Biological Sciences (Shanghai, China). H1975 cells were cultured in RPMI 1640 medium, and A549 cells were cultured in F12K medium. All culture media were supplemented with 10% fetal bovine serum (FBS) at 37℃ in an incubator with 5% CO2. MCM5-Flag, MCM5, and HDAC1 expression plasmids were purchased from Sino Biological (Beijing, China).
Clinical data analysis
The TCGA data were used to analyze the expression of MCM5 and HDAC1 and their effects on the survival of patients with NSCLC. A total of 61 primary NSCLC tissues were collected from patients. The use of these specimens in this study was approved by the Institutional Review Board and the Research Ethics Committee of Tianjin medical university cancer institute and hospital, and written consent was obtained from all participants. Twelve of these specimens were used to analyze the expression of MCM5 through Western blot analysis. All the patients’ samples were divided in accordance with clinical stage and pathological grading. The expression levels of MCM5, HDAC1, and R-loop in these samples were analyzed.
Silver staining
Cells were transfected with flag-MCM5. After 48 h, the cellular extracts were collected and incubated with the anti-FLAG affinity gel (Sigma) for 12 h at 4℃. The eluents of the bed volume were collected and separated through 10% SDS–PAGE. Subsequently, silver staining was performed using the Fast Silver Stain Kit (Beyotime, China).
Immunoprecipitation and Western blot analysis
After transfection, the cell extracts were prepared through incubation in lysis buffer.
For Western blot analysis, the proteins were separated by 10% SDS–PAGE and transferred onto PVDF membranes. The membranes were blocked and incubated with the primary antibodies of MCM5 (ab75975, Abcam, UK), HDAC1 (ab7028, Abcam, UK), and GAPDH (ab8245, Abcam, UK) at 4℃. GAPDH (CAT: 5174, CST, USA) was used as the loading control. After 2 h, the membranes were incubated with a horseradish peroxidase-labeled secondary antibody. Protein expression was visualized using an enhanced chemiluminescence substrate (Millipore). For immunoprecipitation, the cell extracts were centrifuged and incubated with specific primary antibodies at 4℃ overnight with constant rotation. The extracts were incubated with Protein A agarose beads and washed thrice with lysate. After centrifugation and suspension with loading buffer, the denatured protein was boiled for 10 min, separated through 10% SDS–PAGE, transferred onto a PVDF membrane, and detected using a specific antibody.
Colony formation assay
Transfected cells were seeded in 6-well plates at a density of 500 cells per well. The medium was replaced every three days and cultured for 14 days. Then, the clones were fixed with 4% formaldehyde for 10 min and stained with crystal violet. The cell colonies with diameters exceeding 50 μm were counted.
Invasion assay
The Transwell upper chamber filters were coated with Matrigel (BD Biosciences). After transfection, the cells were placed in the upper chamber of the Transwell in serum-free media. The lower chamber media contained 10% FBS. These cells were incubated at 37℃ for 24 h. The noninvasive cells in the top well were removed. Then, the membranes were stained with crystal violet and photographed using a microscope.
Immunohistochemistry
The paraffin-embedded tissue was cut into 4 μm thick sections. After antigen retrieval through heating in microwave oven and blocking with 3% H2O2 solution, the sections were incubated with primary antibodies at 4℃ overnight. The immunohistochemistry staining scores were assessed by two pathologists. The staining score was assessed as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong).
Wound healing assay
The transfected cells were seeded in 24-well plates with 10% FBS culture for 24 h. Then, a linear wound was formed by scraping the cell layer with a 1 μL pipette tip. After the suspended cells were removed with PBS, the culture was allowed to grow by adding a complete medium. Photographs were taken at 0 and 48 h under a microscope.
Scanning electron microscopy (SEM)
The cells were fixed, dehydrated in acetone/isoamyl acetate (1:1), and dried using a gradient concentration of acetonitrile. Gold-coated cells were photographed using a scanning electron microscope (JEOL 6000, Japan).
Molecular docking
The crystal structure of HIF-1α was downloaded from the PDB database and used to perform molecular docking with Rg3 by using the Sybyl X1.1 software.
In vivo experiment
Five-week-old BALB/c mice were purchased from Charles River (Beijing,China), and 24 mice were randomly divided into four groups. Cells stably expressing GFP were transfected with MCM5 or HDAC1 expression plasmid. A total of 5 × 106 cells were inoculated into the lower part of the armpits of BALB/c nude mice. Tumor size and mouse survival were measured every three days. Tumor volume was calculated using the formula: Tumor volume = (length × width2)/2. All animals were euthanized through the intravenous injection of barbiturate at a final concentration of 100 mg/kg. Then, the solid tumors were harvested from the mice by surgery, and the tumor volume was calculated. All tissues were fixed in 4% formalin and embedded in paraffin for H&E histological and immunohistochemical staining. All animal experiments were performed in accordance with relevant ethical standards, and the protocol was approved by the Tianjin Medical University Animal Ethics Committee.
Statistical analysis
All statistical analyses were performed using the GraphPad Prism V.6.0. Comparisons between two groups were performed using the Student’s t-test, whereas comparisons among three or more groups were conducted using ANOVA with Dunnett’s post-test. Differences were considered significant at P < 0.05 and labeled with *.