Supplemantary Material:
Oncogenic CUL4A determines the response to thalidomide treatment in prostate cancer
Shancheng Ren et al.
J Mol Med 2012
Supplementary Methods
Cell lines
The human cancer cell lines were were maintainedat 37°C and 5% CO2 in RPMI 1640, supplemented with 10% FBS. The normal prostate cell linesPWR-1E and RWPE-1 were cultured in keratinocyte serum free medium (GIBICO, 17005-042).
Cell Proliferation and invasion
Cells werealiquoted into a 96-well plate (2,000 per 100 μL/well), incubated for 1 day to 6 days. Cell numbers were determined at each time-point using a Cell Counting Kit-8 according to the manufacturer’s protocol(Dojindo Laboratories). All experiments were done three times.
Invasive potential was measured using an in vitro Boyden chamberassay. Briefly, 5×105 cellsin 0.1 mL serum-free DMEM were added to the well of 8 μm pore membraneBoyden chamber (Costar) coated with Matrigel. The bottomchamber contained keratinocyte serum free medium (GIBICO, 17005-042). Cells were allowed to invade for 48 h and any cells that hadnot penetrated the filters were then removed by scrubbing with cottonswabs. Chambers were fixed for 15min at room temperature with 4%formaldehyde in PBS, stained in 0.1% crystal violet for 20 min, andrinsed in water. Cells that migrated to the bottom surface of the filterwere considered to have invaded through the matrix and were counted.
Soft agar colony formation assay
Stably transfected cells RWPE-1and 22RV1 (10x102) were plated in 6-well culture dishes and incubated in a suspension in 1 ml of DMEM containing 10% calf serum and 0.4% agar on a layer of 1 ml of the same medium containing 0.6% agar. Plates were incubated at 37°C for 2-3 weeks until colonies were formed.
Hoechst staining
The PC-3-Ctrol and PC-3-CUL4Acells were cultured in RPMI 1640 complete medium. After 24 hours of incubation, thalidomide was added for another 48 hours. The cultured medium was removed and the cells were fixed by acetic acid/methanol solution for 10 min. Next, the fixed solution was removed and cells were air-dried for another 10 min. The cell was stained by Hoechst 33258 stain solution (0.5 μg/ml in Hank's balanced salt solution) at room temperature for 30 min. After staining, the solution was removed, the cell was washed three times with distilled water and then one drop of mounting solution was added before being covered by a cover slide. Apoptotic cells showed blue, peripherally clumped or fragmented chromatin.
Retroviral vector construction andvirus transfection
Retroviral vector construction, production and transduction were performed as previously described.
Real-Time PCR Analysis
Total RNA was extracted from celllines and frozen tumor specimens using Trizol reagent (Invitrogen)according to the manufacturer's instructions. CUL4A mRNA expression in prostate cancer cell lines and tissueswas measured by qRT-PCR using an ABI7300 instrument (Applied Biosystems). qRT-PCR was done using a SYBR PrimeScript RT-PCR Kit(Takara) according to the manufacturer's instructions. β-Actin wasused as an internal control. The primers were as follows: CUL4A forward5’-TGCTCCTCATGTTCAACGAG-3’and reverse 5’-TTCTGACGATAGCAGCATCAA-3’and actin forward 5’-CGCGAGAAGATGCCCAGATC-3’ and reverse 5’-TCACCGGAGTCCATCACGA-3’. All experiments were done in triplicate.
Western blot
Cell lyses weresolubilized in RIPA buffer plus protease inhibitors.Fifty-microgram protein extracts were resolved on 8–12% SDS/PAGE,blotted on nitrocellulose, and visualized by immunoblotting with thefollowing primary antibodies: anti–phospho-Ser473 ERK (#4370; Cell SignalingTechnology), anti-total-ERK (#4695; Cell SignalingTechnology), anti-CUL4A (#2699; Cell SignalingTechnology), anti–PARP (#9542; Cell SignalingTechnology), anti-GAPDH (#470661; bioworld).
Apoptosis assay
TdT-mediated dUTP Nick End Labeling (TUNEL) assay was performed using ApopTag Fluorescein In Situ Apoptosis Detection Kit according to the manufacturer’s protocol(Intergen Company, Purchase, NY).
ChromatinImmunoprecipitation(ChIP)Assays
ChIP was carried out essentially as described previously[32]. Solublechromatinwaspreparedandprecipitatedwithanti-triMeK4H3 oracontrolnon-immuneIgG.ForrealtimeChIPqPCR,theSYBRgreenChampionChIPqPCRassaykit(SABiosciences)wasusedand thesampleswereamplifiedwiththeABIPrism7000SequenceDetector(AppliedBiosystems).TheprimersusedforqPCRareasfollows:ERK1:5’- GCCGAACCTCCCGGTGACCT-3’ (Forward), 5’-GGGCCTGGAGCTGTCACGTG-3’ (Reverse). ERK-2: 5’-TGGGTCCTGCTTCATGGGCTAAAT-3’ (Forward), 5’- TTCAAGACTAGCCTGGGCAACACA-3’ (Reverse). Fold enrichment relative to input wascalculated.Theresultsareexpressedasthemean±SDoftriplicatevaluesforeachsample.
- Hung MS, Mao JH, Xu ZD, Yang CT, Yu JS, Harvard C, Lin YC, Bravo DT, Jablons DM, You LA (2011) Cul4A is an oncogene in malignant pleural mesothelioma. J Cell Mol Med 15: 350-358
Supplementay Figure 1. Representative immunohistochemical staining of CUL4A expression in prostate cancers.
Supplementay Figure 2.(a) Low expression of CUL4A in the tumors with shCUL4A. (b) Tumors with shCUL4A exhibit a lower proliferation index Ki-67.
Supplementay Figure 3. CUL4A significantly promoted RWPE-1 invasion, which was abrogated by U0126. Matrigel invasion assay of RWPE-1 cells stably infected with virus expressing XUL4A or control. CUL4A-mediated invasion was abrogated by U0126.
Supplementay Figure 4. Ectopic CUL4A expression in thalidomide resistant rwpe-1 cells increase sensitivity to this treatment.