Cell culture and lentiviral transduction
The human pancreatic cancer cell line Panc-1 was purchased from American Type Culture Collection (ATCC). Panc-1 cells were cultured in Dulbecco's Modified Eagle's Medium (Gibco Laboratory, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS, Gibco) in a humidified atmosphere containing 5% CO2 at 37°C. A knockdown SP1 cell line was established via the transfection of cells with shSP1 lentivirus. The lentiviral plasmids that targeted SP1 (shSP1) were purchased from VigeneBiosciences (Shangdong, China) and the control shRNA sequence (shCtrl) were purchased from Sigma-Aldrich (MerckKGaA, Darmstadt, Germany). For the transduction, a total of 50,000 cells/well were seeded in 6-well plates. The shSP1 lentivirus or negative control lentivirus (shCtrl) was added to the cells in the presence of 5µL Polybrene (Sigma-Aldrich; MerckKGaA, Darmstadt, Germany). After 96 hours, the transduced cells were selected with 1 µg/mL puromycin. Subsequently, the selected cells were treated with different concentrations of Propofol. Importantly, pure propofol was obtained from Sigma Chemical to exclude the influence of lipid emulsion (St Louis, MO, USA).
Cell growth assay
Cell growth was assessed using the thiazolyl blue tetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates (6×103 cells/well) with propofol for 48 hours. MTT solution (Sigma, St. Louis, MO, USA) was added and incubated for four hours at 37°C prior to the precipitation being dissolved in 200 µL dimethyl sulfoxide. The absorption values were measured at 490 nm using a Multiskan Spectrum (Thermo Scientific, USA).
Wound healing assay
In a 6-well plate, Panc-1 cells (1×106 cells/well) were added to DMEM and incubated overnight in order to create a monolayer of cells. A scratch was made in the middle of the well with a pipette tip and the debris was washed away prior to the addition of new media to the wells. Using an optical microscope, the cells were imaged and the initial area of the scratch for the field of view was determined using the area devoid of cells (the length multiplied by the average width, 3 fields of view were measured). The plate was incubated at 37°C for 24 hours, after which the same field of view was imaged and the area devoid of cells was recalculated using the same methodology. The final area of the scratch wound was divided by the initial area, and used to determine the percentage wound remaining of the initial area covered by migrating cells over the 24 hours culture period.
Cell cycle analysis
Cells were incubated with propofol at indicated doses for 24 hours, then washed with cold PBS. Subsequently the cells were fixed using cold 75% ethanol overnight at 4°C, washed with cold PBS and stained in PI for 30 minutes at 37°C prior to being analyzed by flow cytometry.
Total RNA isolation and quantitative real-time PCR
Total RNA was isolated from cell lines using TRIzol (Invitrogen Life Technologies, Carlsbad, USA) according to the manufacturer’s protocol. A NanoDrop spectrophotometer (Thermo Scientific, USA) was used to measure the RNA concentration. Reverse transcription reactions were performed with PrimeScript RT Reagent kit (Takara, Dalian, China) and quantitative real-time PCR (RT-PCR) was performed using FastStart Universal SYBR Green Master (Roche, Mannheim, Germany) and the Step One Plus Real-time PCR system (Applied Biosystems, Singapore). According to the manufacturer's protocol, the thermocycling conditions were as follows: pre-denaturation, 95°C for 30 seconds; amplified reaction, 95°C for 5 seconds, 60°C for 20 seconds, 40 cycles; dissociation curve, 95°C for 60 seconds, 55°C for 30 seconds, 95°C for 30 seconds. The 2−ΔΔCt method was used for relative quantification against the expression levels of β-actin. All experiments were performed in biological triplicates, each with technical triplicates (n = 3). The following primer sequences were used for the aforementioned reactions:
ADAM8: fw: 5′-ACAATGCAGAGTTCCAGATGC-3′,
rev:5′-GGA CCA CAC GGAAGT TGA GTT-3′.
Sp1: fw: 5′-CGGAATTCATGAGCGACCAAGATCACTCCATG‐3′,
GAPDH: fw: 5′-GTC AGT GGT GGA CCTGAC CT-3′,
rev: 5′-TGG TGC TCA GTT TAG CCC AGG-3′.
Cells were lysed using RIPA buffer (Dingguo, Beijing, China) to extract total protein. 50µg of the extracted protein used for 10% SDS-PAGE and subsequently electroblotted on PVDF-membranes. The membranes were incubated with ADAM8 (ab255608, 1:1000, Abcam, Cambridge, UK), SP1(ab231778, 1:1000, Abcam, Cambridge, UK), β-actin (ab8226, 1:1000, Abcam, Cambridge, UK) antibodies overnight at 4°C prior to incubation with goat anti-rabbit HRP-conjugated antibody (ab181662, 1:2000, Abcam, Cambridge, UK). The imaging of proteins was performed using the Oddessy system (Li-Cor biosciences, Lincoln, USA).
Dual-luciferase reporter assays
The transfection and luciferase reporter assay were performed as previously described (14). Wild-type or mutant ADAM8, which contains mutations at the 3'-UTR SP1 binding sites, and a synthesized promoter mimic or vector were co-transfected for 48 hours, then harvested prior to determining the luciferase activity, which was measured using a dual-luciferase reporter assay system (Promega, Fitchburg, WI, USA).
Cell lysates were prepared by using mRIPA buffer (50mM Tris-HCl, pH 7.8, 150mM NaCl, 5mM EDTA, 0.1% Triton-X100, 0.05% NP-40), and total protein was extracted from Panc-1 cells. Subsequently, the lysates were rotated and incubated overnight at 4°C with 2µg of the anti-ADAM8 or anti-SP1, antibody alongside a negative control containing 2µg of a rabbit IgG antibody, supernatant without any antibody (Input) was used as a positive control. After incubation, the mixture was incubated with Protein A or G Sepharose agarose beads at 4°C for 3 hours. The beads were collected and sequentially washed five times with RIPA lysis buffer (1ml), then analyzed by western blotting using anti-ADAM8 or anti-SP1 antibody correspondingly. The intensity of the specific bands was estimated by Image J2X software package. The assays were repeated at least three times.
All values were presented as mean from three independent experiments ± standard deviation (SD). SSPS 20.0 software was used for statistical analysis. A one-way ANOVA followed by Duncan's multiple range test and Student’s t test were performed to assess variation among experimental groups, the threshold for significance was set at P < 0.05.