RNA Extraction and Semiquantitative RT-PCR
Total RNA was extracted from cultured cells using Trizol reagent (Invitrogen) accordingto the manufacturer’s instructions. Extracted RNA wastreated with DNase (Fermentas, Vilnius, Lithuania) toremove DNA contamination. For cDNA synthesis, 2μgof total RNA was reverse transcribted using ReverAidTMFirst Strand cDNA Synthesis Kit (Fermentas). PCR wasperformedwithExTaq (Takara, Japan). The primer sequencesand the size of amplified product were as follows:
CUL4A (225bp): 5′-GACAACAATGAGAACCTTCAGGAGA-3′(forward)
5′-CTGGCGCCGGTTACAGAACCA-3′ (reverse)
GAPDH (613bp): 5′-GGCATCGTGATGGACTCCG-3′ (forward)
5′-GCTGGAAGGTGGACAGCGA-3′ (reverse)
p53 (200bp): 5′-TGTGGACCTCAGGTTGGACT -3′(forward)
5′-CTTCTGCAGGGC TTTCATGT -3′ (reverse)
p21 (250bp): 5′-ATGGATCTGACCTCGGAGTG-3′(forward)
5′-CCTCACATAAGAGCGCATCA-3′(reverse)
p27 (540bp): 5′-GTGGGGCGCCCCAGGCACCA-3′ (forward)
5′-CTTCCTTAATGTCACGCACGATTTC-3′ (reverse)
GAPDH was used as an internal control. The PCRproducts were separated on 1.5% agarose gels by electrophoresis.The intensity of bands was determined usingthe Image-Pro Plus 6.0 software.
Western blot analysis
Cells were washed with cold PBS and lysedin ice-cold RIPA buffer containing protein inhibitors.Cell lysates were incubated on ice for 30 min and thencentrifuged at 4℃ for 10 min. Supernatants were collected,and protein concentrations were measured.Equivalent amounts of protein in each sample were separatedon a 10% SDS-PAGE gel for separation and thenelectrotransferred to PVDF membrane. Membraneswere blocked and then probed with primary antibodiesfollowed by the horseradish peroxidase (HRP)-conjugatedgoat anti-mouse or rabbit IgG antibodies.The primaryantibodies used were as follows: rabbit polyclonalanti-CUL4A antibody (Santa Cruz Biotechnology), mousemonoclonal anti-p53 antibody (Santa Cruz Biotechnology),mouse monoclonal anti-p21 antibody (ChemiconInternational Inc., Temecula, CA), mouse monoclonalanti-p27 antibody (Cell Signaling Tech., Beverly, MA),and rabbit monoclonal anti-Bax antibody (Cell Signaling Tech.). Mouse monoclonal antiβ-actin antibody (Sigma, St. Louis, MO) was used as aninternal control. The membranes were developed usingan ECL detection system (Pierce, Rockford, IL). The intensityof bands was determined using the Image-ProPlus 6.0 software. The Western blot experiments wererepeated at least three times.
Confocalimmunofluorescence microscopy
Cells were plated on culture slides (Costar, Manassas, VA, USA). After 24 hrs, the cellswere rinsed with phosphatebufferedsaline (PBS) and fixed with 4% paraformaldehyde in PBS, and cell membrane was permeabilizedusing 0.5% Triton X-100. These cells were then blocked for 30 min in 10% BSA (Sigma, Aldrich St. Louis, MO, USA) in PBS and then incubated with primary monoclonal antibodies in 10% BSA overnight at 4℃. After three washes in PBS, the slides were incubated for 1 hour in the dark with FITC-conjugated secondary goat anti-mouse, or goat anti-rabbit antibodies(Invitrogen, Grand Island, NY, USA). After three further washes, the slides were stained with 4-,6-diamidino-2-phenylindole (DAPI; Sigma, Aldrich St. Louis, MO, USA) for 5 min to visualize the nuclei, and examined using an Carl Zeissconfocal imaging system (LSM 780) ( Carl Zeiss, Jena, Germany).
Immunohistochemistry
For immunohistochemistry, 5-μm thick tissue sectionswere cut fromparaffin-embedded samples. Immunostainingwas performed following standard methods.The sections were incubated with primary anti-CUL4A and p53 antibody at 4℃ overnight, followed by staining withABC kit (Santa Cruz Biotechnology). The sections werecounterstained with hematoxylin. In negativecontrols, all the incubation conditions described earlierwere followed except that the primary antibody wasreplaced by phosphate-buffered saline (PBS). The stainingprocedures were repeated at least three times.
Cell proliferation assay
Cells were seededin 96-well plates in triplicate at densities of 1×103 per well. Cell proliferation was monitored at 12, 24, 48, 72, and 96 hrs using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)(Sigma, St. Louis, MO, USA). In brief, the MTT assay was performed by adding 20 μl MTT (5 mg/ml) for 4 hrs. Light absorbance of the solution was measured at 570nm on a microplate reader (PerkinElmer, Waltham,Massachusetts, USA).
Colony formation assay
Cells were seeded in triplicate at 500 cells/6-cm dishes in complete medium. After 3 weeks of growth, the cells were fixed and stained with crystal violet stain (0.1%, w/v in 20 nM 4-morpholinepropanesulfonic acid; Sigma, St Louis, MO, USA), and the numbers of colonies containing morethan 50 cells were counted. All experiments were repeated at least three times. Plating efficiency was determined as the number of colonies formed divided by the total number of cells plated.
Cell invasion and motility assay
Invasion of cells was measured in Matrigel (BD, Franklin Lakes, NJ, USA) -coated Transwell inserts (6.5mm, Costar, Manassas, VA, USA) containing polycarbonate filters with 8-μm pores. The inserts were coated with 50μl of 1mg/ml Matrigel matrix according to the manufacturer’s recommendations. 2×105cells in 200μl of serum-free medium were plated in the upper chamber, whereas 600μl of medium with 10% fatal bovine serum were added to lower well. After 24hrs incubation, cells that migrated to the lower surface of the membrane were fixed and stained. For each membrane, five random fields were counted at ×10 magnification. Motility assays were similar to Matrigel invasion assay except that the Transwell insert was not coated with Matrigel.