Cell lines, reagents and antibodies
Human lung cancer cell lines A549, 95D and H1229 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured either in in F-12K medium (A549 cells) or RPMI-1640 medium (95D cells and H1229 cells) containing 10% fetal bovine serum, at 37°C with 5% v/v CO2. MTT assay reagents were purchased from DingGuo Biotech (Beijing, China). 5-Bromo-2'-deoxyuridine (BrdU) assay reagents were purchased from Chemicon International (Temecula, CA, USA). Anti-iASPP mAb used for Western blot assay was purchased from Abcam (Boston, MA, USA). Anti-iASPP rAb using for Immunohistochemical assay was purchased from Rockland Immunochemicals, Inc., (Gilbertsville, PA, USA). Anti-GAPDH monoclonal was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Lentivirus-mediated shRNA delivery
Sequences of iASPP shRNA were inserted into the pGCL-GFP lentivirus RNAi expression system. The shRNA containing vectors were transfected together into 293T cells with pHelper1.0 and the lentiviral helper plasmid pHelper2.0 to generate the respective lentiviruses. Viral stocks were collected from the transduced 293T cells and were used to infect A549 cells, 95D cells and H1229 cells. The sequence of iASPP nonsense shRNA was: AATGTACTGCGCGTGGAGA; the sequence of iASPP shRNA was AACACATGGATCTGAAGCAGA. The mRNA and protein levels were measured 72 hrs after cells being infected.
Quantitative RT-PCR analysis of iASPP expression
Total RNA was extracted and reverse transcribed into cDNA using M-MLV-RTase (Promega, Madison, WI, USA). The resulting cDNA was used for PCR using the SYBR-Green Master PCR Mix (Applied Biosystem, Carlsbad, CA, USA) in triplicates. Primers for qRT-PCR were as follows: iASPP forward primer: GGCGGTGAAGGAGATGAAC; iASPP reverse primer: TGATGAGGAAATCCACGATAGAGA; p53 forward primer: CCTCCTCAGCATCTTATCC; p53 reverse primer: ACAAACACGCACCTCAAA; p21 forward primer: GGGACAGCAGAGGAAGACC; p21 reverse primer: GACTAAGGCAGAAGATGTAGAGC; PUMA forward primer: GACGACCTCAACGCACAG; PUMA reverse primer: CACCTAATTGGGCTCCATCTC. PCR and data collection were performed on the TP800 qPCR System (Takara, Japan). All quantitations were normalized to an endogenous β-actin control. β-actin forward primer: GGCGGCACCACCATGTACCCT; β-actin reverse primer: AGGGGCCGGACTCGTCATACT. The relative quantitation value for each target gene compared to the calibrator for that target is expressed as 2-(Ct-Cc) (Ct and Cc are the mean threshold cycle differences after normalizing to β-actin).
Western blot
Protein samples prepared from the cells were subjected to SDS-PAGE, transferred to PVDF membranes (Millipore, Kankakee, IL, USA) and detected with appropriate primary antibodies followed by horseradish peroxidase-conjugated goat, anti-mouse or rabbit IgG. The protein signals were detected using SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL, USA).
MTT assay
All the cells, including those transfected, were grown in exponential phase and detached by trypsin/EDTA treatment. Viable cells (2,000 cells/ml) were plated into 96-well tissue culture plates (100 μl complete medium/well) and cultured at 37°C in 5% CO2. At different time points, MTT reagent was added (10 μl per well) and incubated at 37°C for 4 hr. The reaction was stopped with addition of 100 μl DMSO and the optical density was determined at OD570 nm on a multi-well plate reader. Data from three independent experiments were analyzed by student t test and p < 0.05 was considered statistically significant.
BrdU assay
Cells were seeded into 96-well plates (1,500 cells/well) and cultured at 37°C in 5% CO2. At different time points, BrdU reagent was added (20 μl/well) and incubated at 37°C for 4 hr. Cells were then fixed in a fixation solution for 30 min. After washing three times with a washing buffer, anti-BrdU antibody was added (50 μl/well) and incubated at 37°C for 1 hr. Following washing, an enzyme conjugated secondary antibody was added (50 μl/well) and incubated at 37°C for a further 30 min. Colour was then developed by incubation with 50 μl TMB substrate for 30 min in dark and the optical density was determined at OD490 nm on a multi-well plate reader. Data from three independent experiments were analyzed by student t test and p < 0.05 was considered statistically significant.
Colony formation assay
Cells were seeded into six-well plates (200 cells/well) (in three duplicate wells) and cultured at 37°C in 5% CO2. After two weeks, the cells were fixed with paraformaldehyde for 30 min and then stained with GIEMSA for 10 min. ddH2O was used to wash the cells three times to obtain a clean background. The number of colonies and the cell number in each colony were counted and statistically analyzed.
Immunohistochemical Staining
Tissues sections (5-μm thick) were dewaxed, followed by quenching the endogenous peroxidase with 3% H2O2 in methanol for 30 min. Prior to staining, non-specific binding was blocked by incubation with 10% BSA in PBS at 37°C for 1 hr. Tissue sections were incubated with pre-immune IgG or specific antibodies in PBS containing 1% BSA at 4°C overnight, followed by incubation with a horseradish peroxidase-conjugated anti-mouse or rabbit antibody. Colour was then developed by incubation with an ImmunoPure Metal Enhanced Diaminobenzidine (DAB) Substrate kit (Pierce). After each incubation, tissue sections were washed three times in PBS for 10 min. Tissue sections were finally counterstained with hematoxylin. For determination of iASPP immunoreactivity, cytosolic staining of yellowish or brownish granules was graded as follows: 0 for background staining, 1 for faint staining, 2 for moderate staining and 3 for strong staining. In addition, positive staining areas in entire tissue section were graded as follows: 0 for <5%, 1 for 5-25%, 2 for 26-50%, 3 for 51-75%, and 4 for 76%-100%. When combining these two parameters, 0-2 and ≥3 were considered negative and positive staining, respectively.
Statistical analysis was carried using SPSS (version 16). Fisher's Exact test was used for analyzing the immunohistochemical data and Student t test for other quantitative data.