Three human lung adenocarcinoma cell lines, A549, A2, and H1299; two human large cell lung carcinoma cell lines, 95C and H460; and one small cell lung cancer cell line, H446, were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Los Angeles, CA, USA). Human embryo lung fibroblasts (MRC5) were maintained in DMEM supplemented with 10% FBS. The cells were maintained at 37°C in a humidified chamber containing 5% CO2 and 95% air.
CypA RNAi lentivirus generation
Four CypA-targeting oligonucleotides serving as RNAi candidates were designed based on the full-length human CypA cDNA sequence and cloned into the pGCsi-H1/Neo/GFP vector (Shanghai Genechem Co. Ltd., Shanghai, China). CypA-Si2 (CTGACTGTGGACAACTCGAAT), which matches the sequence located at nucleotides 559–579 of the CypA cDNA, proved to be the most effective at decreasing the CypA mRNA level and was used to knock down endogenous CypA in the following experiments. A nonsilencing-siRNA (NS-siRNA, TTCTCCGAACGTGTCACGT) was used as a negative control. Oligonucleotides encoding CypA-Si2 or NS-siRNA together with a loop separating the complementary sequences were synthesized and inserted into the pGCL-GFP lentivirus construct, which contained an H1 promoter and an ampicillin resistant cassette (Shanghai Genechem Co. Ltd.). The recombinant virus was packaged using a Lentivector Expression System (Shanghai Genechem Co. Ltd.), according to the manufacturer’s instructions.
Recombinant lentiviral particle infection of target cells
Target cells were plated into 96-well culture plates at 5,000/well. Cells were infected with recombinant virus carrying CypA-siRNA or NS-siRNA 24 h later. GFP expression was detected via fluorescence microscopy (Nikon, Tokyo, Japan) to determine the infection efficiency. Cells were cultured for an additional 2 weeks prior to harvest, at which time CypA expression was assessed by quantitative real-time PCR (qRT-PCR) and Western blot analysis.
Reverse transcription PCR and qRT-PCR analyses
Total RNA was extracted using TRIzol Reagent (Invitrogen) with the RNA quality being assessed by formaldehyde-agarose gel electrophoresis. First-strand cDNA was obtained by reverse-transcription using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA) as instructed by the manufacturer. qRT-PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in a total volume of 25 μL with 2 μL of cDNA, and detected using an ABI7500 Real-Time PCR System (Applied Biosystems). The real-time PCR conditions for CypA and β-actin were: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s with the primers CypA sense (5’-CATACGGGTCCTGGCATCT-3’), CypA antisense (5’-TGCTGGTCTTGCCATTCC-3’), β-actin sense (5’-TTAGTTGCGTTACACCCTTTC-3’), and β-actin antisense (5’-GCTGTCACCTTCACCGTTC-3’). β-actin was used as internal loading controls. Relative mRNA levels are presented as 2-ΔCT. Three independent experiments were completed; each reaction was performed in triplicate. All data are shown as means ± SEM. A no-template control (NTC) was used to avoid genomic DNA contamination.
Western blot analysis
Whole-cell lysates were harvested in ice-cold lysis buffer (10 mM Tris–HCl, pH 7.4, 1 mM EDTA, 0.1% Triton X-100, and 0.1% SDS) containing protease inhibitors (2 μg/mL aprotinin, 10 μg/mL antipain, 2 μg/mL pepstatin, and 2 mM benzamide). After the removal of cell debris by centrifugation (12,000 × g, 10 min), the protein concentration in the supernatants was measured using bicinchoninic acid protein assay reagent (Pierce Chemical Co., Rockford, IL, USA) according to the manufacturer’s instructions. Ten micrograms of total protein were subjected to SDS-PAGE and wet-transferred to nitrocellulose membranes. The membranes were probed with anti-human CypA polyclonal rabbit serum (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:500 dilution and anti-GAPDH antibodies (CoWin Biotech, Shanghai, China) diluted 1:5,000 in 5% (w/v) nonfat dry milk in TBST (50 mM Tris–HCl, 138 mM NaCl, and 0.1% Tween-20, pH 7.6). Secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology) were diluted 1:5,000. Signals were detected using the SuperSignal West Femto Chemiluminescent Detection System (Pierce Chemical Co.) and exposed to Kodak X-OMAT film.
For the detection of ERK1/2, p38, JAK2, and STAT5, rabbit anti-ERK1/2 antibody (Abcam, Cambridge, England, UK), mouse anti-pERK antibody (Abcam, Cambridge, England, UK), rabbit anti-p38 antibody (Abcam, Cambridge, England, UK), mouse anti-pp38 antibody (Abcam, Cambridge, England, UK), mouse anti-JAK2 antibody (Abcam, Cambridge, England, UK), rabbit anti-pJAK2 antibody (Abcam, Cambridge, England, UK), rabbit anti-STAT5 antibody (Abcam, Cambridge, England, UK), and mouse anti-pSTAT5 antibody (Abcam, Cambridge, England, UK) were used.
High-content cell cycle analysis
The Cellomics ArrayScan HCS Reader was used to quantify cell parameters by immunofluorescence staining. In brief, cells were cultured in 96-well plates at 37°C for 24hours, washed three times with phosphate-buffered saline, and then fixed in 4% paraformaldehyde for 10 min followed by 0.2% TritonX-100 for 15 min at room temperature. After blocking of non-specific binding sites in 3% BSA for 30 min, cells were incubated with anti-CypA primary antibodies overnight at −4°C and treated with anti-rabbit IgG-TRITC secondary antibody for 30 min at room temperature. The expression level of CypA protein was measured with the Cellomics ArrayScan HCS Reader using the ArrayScanTM software.
Cell proliferation assay
Tumor cells (3,000/well) were seeded in flat-bottom 96-well plates. The next day, the cells were serum-starved for 24 h and exposed to 0.2% BSA. Cell proliferation was evaluated by a 3-(4, 5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS; Promega) assay, which was performed at a fixed time every day for the next 5 days. A total of 20 μL of MTS was added to each well, followed by incubation for 3 h. The absorbance was recorded at 490 nm with an EL-800 universal microplate reader (Bio-Tek Instruments, Winooski, VT, USA). This assay was repeated three times in triplicate. Cell doubling-time was calculated using doubling time online calculator .
Colony formation assay
Three-hundred cells were suspended in 2 mL of culture medium and seeded in 6-well plates. The cells were maintained for 10 days with a change of media every 3 to 4 days. The number of colonies with >50 cells in each well was counted on the 10th day. The colonies were visualized and counted by the trypan blue exclusion method. The assay was repeated three times in triplicate.
Anchorage-independent growth assay
A total of 1,000 cells were resuspended in 1 mL of 0.6% agarose in 6-well plates coated with a 1.2% agarose bed. Triplicate cultures of each cell type were maintained for 21 days; the medium was changed every 7 days. The number of colonies >50 μm (~100 cells) in diameter per well was counted manually with the aid of Alpha View Analysis Tools (Alpha Innotech Corp., San Leandro, CA, USA). All experiments were performed in triplicate and repeated three times.
Cell migration assay
Cell motility was assessed by two assays. For the wound healing assay, confluent cell monolayers were wounded with a sterile pipette tip and cultured in serum-free medium in 6-well plates. The wounds were observed at 0, 12, and 24 h along the scratch, and representative images of fixed positions were acquired with a phase-contrast microscope. The wound areas were measured using Alpha View Analysis Tools, and the percentage wound closure was determined.
A migration assay was performed in a 24-well Transwell unit containing an 8-μm pore size polycarbonate membrane (Costar, Cambridge, NY, USA) as reported previously . After starvation for 12 h, the cells were suspended and plated in the upper compartment with serum-free medium. The lower compartment was filled with medium containing 10% FBS for use as a chemoattractant. After 24 h,the cells in the upper compartment were removed completely by gentle swabbing. Cells migrating to the lower surface of the membrane were determined using crystal violet. The number of cells on the lower surface of the membrane was counted in five microscopic fields at 200× magnification. Triplicate samples were tested. The data are presented as means ± SEM.
Cell invasion assay
The invasion assay was determined by transwell chamber as reported previously . Briefly, cells were starved for 12 h, suspended, and seeded in the up-per compartment on Matrigel Matrix (5 μg/mL; BD Pharmingen, San Diego, CA, USA)-coated 24-well Transwell units (Costar). RPMI 1640 medium supplemented with 10% FBS was added to the lower compartment for use as a chemoattractant. After incubation for 24h and 48 h respectively, cells attached to the lower surface of the membrane were stained by crystal violet. The number of cells on the lower surface of the membrane was counted in five microscopic fields at 400× magnification. Triplicate samples were assayed. The data are presented as means ± SEM.
Gelatinolytic activity and quantity in conditioned media were analyzed by gelatin zymography. In brief, serum-free conditioned medium was centrifuged to remove cellular debris and then subjected to non-reducing SDS-PAGE using an 8% separating gel containing 0.1% gelatin. Subsequently, the gels were washed twice in 2.5% Triton X-100 for 30 min at room temperature to remove SDS and incubated in reaction buffer (50 mM Tris, 0.2 M NaCl, and 5 mM CaCl2) for 48 h at 37°C to hydrolyze the copolymerized protein substrate in a zone around their electrophoresed position. The gels were subsequently stained with 0.5% Coomassie brilliant blue R-250 to visualize the digested areas as clear bands against a blue background of undegraded gelatin. The gels were then scanned and analyzed using Alpha View Analysis Tools.
CypA inhibitor 239836 function studies
Cell proliferation assay and gelatinolytic activity was also assessed in the presence of CypA inhibitor 239836(Merck, Darmstadt, Germany). In brief, A549 and 95C cells were seeded onto 96-well plates or 6-well plates. 239836 (0, 0.1, 1, and 10 μg/mL) was added 24 hours later. The vehicle (DMSO, 0.3 μl/ml) was used as a control. Cell growth curve and the activity of MMP9 were detected.
The data are presented as the means ± SEM of at least three independent experiments. Statistical analysis was performed using Student’s t-test. Unless otherwise indicated, P<0.05 was deemed significant.